Jo-Ann M. Jablonski, Andrew J. AubinWaters Corporation, Milford, MA, USA

Figure 1. TIC of the mass-directed collection of the three compounds (5 mg/mL each in DMSO) in the Preparative Chromatography Mix Standard; 1 Diphenhydramine (m/z = 256.2, Fractions 22 and 23), 2 Flavone (m/z = 223.2, Fraction 24), and 3 Diclofenac (m/z = 296.0, Fraction 25).

Time2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

5

1 2

3

22

24

25

23

XBridge BEH C18 OBD Prep, 19 x 50 mm, 5 µm Mobile phase A: 0.1% Formic Acid in water Mobile phase B: 0.1% Formic Acid in acetonitrileSystem: AutoPurificationDetection: Mass, with ACQUITY QDa Detector

Injection volume: 171 µL Flow rate: 25 mL/min

Gradient: Time %A %B0.0 95 5 6.0 5 95 7.0 5 95 7.1 95 5 10.0 95 5

GOA L

To provide chromatographic method conditions

for preparative systems configured with an

ACQUITY® QDa® Detector using the Preparative

Chromatography Mix Standard as a system test

prior to injecting samples for isolation.

BAC KG ROU N D

Compound isolation requires careful

chromatographic system preparation to ensure

that the instrumentation is properly setup prior

to injecting valuable samples. The Preparative

Chromatography Mix Standard is specially

formulated with an acid, a base, and a neutral

compound and can be used daily to evaluate

the condition of the column and to benchmark

system performance. Changes in column

efficiency or collection parameters are apparent

and can be addressed before sample loss

occurs. In this technology brief, we document

the use of this standard mixture in the Waters®

AutoPurification™ System configured with

an ACQUITY QDa Detector.

■■ Preparative Chromatography Mix Standard,

Part Number 186006703

■■ Preparative Column: XBridge® BEH C18

OBD Prep, 19 x 50 mm, 5 µm,

Part Number 186002977

■■ Analytical Column: XBridge BEH C18,

4.6 x 50 mm, 5 µm,

Part Number 186003113

The Preparative Chromatography Mix Standard assesses the suitability of the preparative LC system for isolating target compounds, ensuring proper system setup and column performance before purifying valuable samples.

Typical Conditions for Analyzing and Isolating the Compounds in the Preparative Chromatography Mixture Standard with an ACQUITY QDa Detector

R E SU LT S

The separation shown in the TIC chromatogram in Figure 1 is typical for injections

of the prep standard on the XBridge BEH C18 Column. While diphenhydramine, a

base, elutes first at about 2.5 minutes, flavone and diclofenac, a neutral compound

and an acidic compound, respectively, elute later but with good resolution between

them. Different method conditions and column chemistries may change the

chromatographic profile, but routine use of the prep standard should show

consistent results when the parameters are held constant.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Analysis of the collected fractions, shown in Figure 2,

indicates that the isolated compounds are reasonably

pure. These results suggest that the chromatographic

system is properly set up and is performing as

expected, making it acceptable for isolating and

purifying compounds. The ACQUITY QDa Detector

method parameters are the same at both the analytical

and preparative scales, and are listed in Figure 3.

SUMMA RY

■■ The Preparative Chromatography Mix Standard

is suitable for assessing the chromatographic

performance and system suitability before

injecting samples for isolation and purification

on prep systems configured with an

ACQUITY QDa Detector.

■■ While different column chemistries and method

conditions may alter the chromatographic profile

of the Preparative Chromatography Mix Standard,

repeatability of results from day to day is a good

indication of the prep system’s suitability for

isolating compounds.

■■ The mass spectrometer method parameters for

the ACQUITY QDa Detector illustrated here are

acceptable for this sample mixture, but should

be optimized for other isolations. Time2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00

%

8

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00

%

3

2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00

%

3

Fr 22 DiphenhydramineBase

Fr 24 FlavoneNeutral

Fr 25 DiclofenacAcid

XBridge BEH C18, 4.6 x 50 mm, 5 µm

Mobile phase A: 0.1% Formic Acid in water Mobile phase B: 0.1% Formic Acid in acetonitrile

System: AutoPurificationDetection: Mass, with ACQUITY QDa Detector

Injection volume: 20 µL Flow rate: 1.46 mL/min

Gradient: Time %A %B

0.0 95 5 6.0 5 95 7.0 5 95 7.1 95 5 10.0 95 5

Figure 2. TICs of the fraction analysis of the three collected compounds in the Preparative Chromatography Mix Standard. Fraction 23 was not analyzed due to the very low fraction volume.

ACQUITY QDa Detector

Ionization mode: ES+ Capillary voltage: 0.8

Data: Centroid Probe temperature: 600

Mass range: 100 – 650 Detector gain: 1

Cone voltage: 15 Makeup: 90% water/

10% acetonitrile/0.01% formic acidSampling frequency: 5 Hz

Figure 3. ACQUITY QDa Detector method parameters.

Waters, ACQUITY, QDa, XBridge, and The Science of What’s Possible are a registered trademarks of Waters Corporation. AutoPurification is a registered trademark of Waters Corporation. All other trademarks are the property of their respective owners.

©2014 Waters Corporation. Produced in the U.S.A. July 2014 720005105EN LM-PDF