Discovery^® HPLC Columns for Fast LC-MS Analysis

LC-MS Columns

The Discovery LC-MS line offers short length columns for fast LC-MS analysis that are rugged and dependable. Supelco offers a robust product that withstands column shock due to fast gradient turnaround times. Columns installed on LC-MS instruments are typically operated using gradients of 5%-95% organic with gradient cycle times of 2-3 minutes. Our column lifetime is maximized through the use of stable bonding chemistry, high purity silica and optimal proprietary packing procedures.

The Discovery line of HPLC columns offers excellent retentivity, selectivity and reproducibility for optimal results. LC-MS formats are now available with Discovery C18, C8, Cyano and HS F5 phases.

Discovery LC-MS Columns Deliver:

• High speed separations
• Excellent peak shape
• Demonstrated ruggedness, stability and reproducibility
• Excellent LC-MS bleed characteristics

Properties		C18		C8
Bonded Phase:		Octadecylsilane, endcapped		Octylsilane, endcapped
Silica:		Spherical, high purity, <10 ppm metals		Spherical, high purity, <10 ppm metals
Particle Size:		5 µM		5 µM
Pore Size:		180 Å		180 Å
Surface Area:		200 m2/g		200 m2/g
%C:		12%		7.5%
Coverage:		3 µmol/m2		3.4 µmol/m2
Properties		CYANO		HS F5
Bonded Phase:		Cyanopropylsilane, endcapped		Pentafluorophenylpropyl, endcapped
Silica:		Spherical, high purity, <10 ppm metals		Spherical, high purity, <10 ppm metals
Particle Size:		5 µM		3.5 µM
Pore Size:		180Å		120Å
Surface Area:		200 m2/g		300 m2/g
%C:		4.5%		12%
Coverage:		3.5 µmol/m2		4 µmol/m2

High Speed Separations

Discovery LC-MS columns can shorten analysis times to less than 4 minutes for the study of antibiotics.

Figure 1. Rifampicin on Discovery C18

Column 1:	Discovery C18, 15 cm x 2.1 mm ID, 5 µM particles
Cat. No.:	50495521
Column 2:	Discovery C18, 2 cm x 2.1 mm ID, 5 µM particles
Cat. No.:	577507-U
Mobile Phase:	60:40, 33 mM HCO2H:CH3CN
Flow Rate:	0.2 mL/min
Temp.:	35 ºC
Det.:	ESI(+), full scan
Inj.1:	6 µL
Inj.2:	0.8 µL
Sample:	10 mg/L in 50% CH3OH

Figure 2. Application of Short, Fast LC-MS Columns for Analysis of Veterinary Antibiotics on Discovery C18 and C8 Columns

Column 1: Discovery C18, 2 cm x 2.1 mm ID, 5 µM particles Cat. No.: 577507-U Column 2: Discovery C8, 2 cm x 2.1 mm ID, 5 µM particles Cat. No.: 577501-U Mobile Phase: A=50mM HOAc/NH4OH, (pH 6.0), B=water, C=CH3OH Flow Rate: 0.2 mL/min Temp.: 65 ºC Inj.: 1 µL Det.: ESI(-) Sample: 1. Lasalocid A (1 µg/mL) 2.3. Salinomycin (10 µg/mL) 4. Narasin (5 µg/mL)

Discovery C18, A:B:C = 20:5:75

Discovery C8, A:B:C = 20:10:70

Column 1:	Discovery C18, 2 cm x 2.1 mm ID, 5 µM particles
Cat. No.:	577507-U
Column 2:	Discovery C8, 2 cm x 2.1 mm ID, 5 µM particles
Cat. No.:	577501-U
Column 3:	Discovery Cyano, 3 cm x 2.1 mm ID, 5 µM particles
Cat. No.:	577514-U
Mobile Phase:	A=50 mM HCO2H, B=water, C=90:10 CH3OH:CH3CN
Flow Rate:	0.2 mL/min
Temp.:	35 ºC
Inj.:	1 µL
Det.:	ESI(+)
Sample:
1.	Erythromycin c (1 µg/mL)
2.	Clarithromycin (1 µg/mL)

Discovery C18
A:B:C = 20:35:45

Discovery C8
A:B:C = 20:35:45

Discovery Cyano
A:B:C = 20:40:40

Mechanical Stability via Fast Gradient Testing

The Supelco Discovery LC-MS HPLC columns are rigorously tested in order to provide the highest mechanical stability under repeated gradient cycles. Table 1 shows of the impact on column efficiency, peak shape, retention times and back pressure after 1000 cycles of a 2-minute rapid (ballistic) gradient with 1-minute equilibration period.

There is no loss of column efficiency or increase in tailing or column backpressure, and no change in retention time. See Table 2 for details of testing.

Table 1. Rapid Gradient Turnaround Testing of Discovery LC-MS HPLC Columns

Phase	Efficiency (N/column)	USP Tailing Factor	Retention Time (min.)	Pressure (bar)
Discovery C18 (3 cm x 3.0 mm ID)
Before stability test	2792	1.04	1.989	39
After stability test	2899	1.09	1.985	41
Discovery C8 (2 cm x 2.1 mm ID)
Before stability test	1451	1.05	1.767	26
After stability test	1507	1.09	1.761	25
Discovery Cyano (3 cm x 4.6 mm ID)
Before stability test	3329	1.01	1.368	44
After stability test	3448	1.01	1.362	40

Table 2. Test Conditions

Discovery C18 (3 cm x 3.0 mm ID)
Flow:		0.8 mL/min
Gradient:		10-90% methanol over 2 minutes back to 10% for 1 minute equilibrium for each 3 minute cycle
Temp.:		30 °C
Wavelength:		254 nm
Pressure:		76 bar for first cycle 75 bar after last cycle
No. of Cycles:		1,135 three-minute
Total Volume:		2724 mL
Void Volume:		0.447
Discovery C8 (2 cm x 2.1 mm ID)
Flow:		0.5 mL/min
Gradient:		10-90% methanol over 2 minutes back to 10% for 1 minute equilibrium for each 3 minute cycle
Temp.:		30 °C
Wavelength:		254 nm
Pressure:		40 bar after first cycle 38 bar after last cycle
No. of Cycles:		1,089 three-minute
Total Volume:		1634 mL
Void Volume:		0.393
Discovery Cyano (3 cm x 4.6 mm ID)
Flow:		0.2 mL/min
Gradient:		10-50% acetnonitrile over 2 minutes back to 10% for 1 minute equilibrium for each 3 minute cycle
Temp.:		30 °C
Wavelength:		254 nm
Pressure:		72 bar after first cycle 70 bar after last cycle
No. of Cycles:		937 three-minute
Total Volume:		5622 mL
Void Volume:		0.434

Discovery C18 Bleed Analysis

Mass spectral responses from background ions often inhibit both qualitative and quantitative analysis in LC-MS experiments. Common sources of background ions include the solvents, interface or system contamination, and HPLC column bleed. The presence of background ions can be reduced by using high quality solvents such as LC-MS, maintaining a clean LC-MS system, and by using high quality HPLC stationary phases from Supelco.

Supelco utilizes technologically advanced bonding chemistry and highly purified silica to minimize the potential for column bleed.

LC-MS Bleed Analysis

The Total Ion Chromatogram (TIC) shown in the top of Figure 4 shows the result from an MS detector of one gradient cycle with a Discovery C18, 2 cm x 2.0 mm ID column in-line. The two selective ion chromatograms (SIC) in the bottom of Figure 4 show the mass spectrum taken from the shaded region of the TIC (at the high organic content point in the gradient). The bottom right SIC shows that the Discovery C18 column does not introduce any additional masses that were not in the blank run (bottom left SIC). The Discovery C18 column exhibits low bleed for LC-MS analyses.

Total Ion Chromatogram

Discovery C18, 2 cm x 2.0 mm ID, acetonitrile gradient in presence of 0.1% formic acid / 5 cycles / ESI (+) mode acquisition

Figure 4. LC-MS Bleed Study.

Blank Run (no LC column) (19-21 min.)

Discovery C18 column (19-21 min.)

Ordering Information

Phase	ID (mm)	Length (cm)	Product. No.
5 µM Discovery C8 Columns
	2.1	2	577501-U
	2.1	3	577502-U
	3.0	2	577503-U
	3.0	3	577504-U
	4.6	2	577505-U
	4.6	3	577506-U
5 µM Discovery C18 Columns
	2.1	2	577507-U
	2.1	3	577508-U
	3.0	2	577509-U
	3.0	3	577510-U
	4.6	2	577511-U
	4.6	3	577512-U
5 µM Discovery Cyano Columns
	2.1	2	577513-U
	2.1	3	577514-U
	3.0	2	577515-U
	3.0	3	577516-U
	4.6	2	577517-U
	4.6	3	577518-U
3 µM Discovery HS F5 Columns
	2.1	3.3	567501-U
	3.0	3.3	567505-U
	4.6	3.3	567509-U

Related Literature

• Application Report 46 - Drugs of Abuse on Discovery HS F5 (T003046)
• Application Report 134 - Separation of Dopamine and Related Compounds using Discovery HS F5 with Mass Spectrometric Detection (T004134)
• Application Report 136 - Resolution of Bilirubin and Biliverdin on Discovery C18 (T004136) [Note: With MS compatible mobile phase.]
• 2004 Poster Presentation - Advantages of Polar, Reversed-Phase HPLC Columns for the Analysis of Drug Metabolites (T404045)

How to Contact Supelco

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Phone: 800-247-6628 or 814-359-3441
fax: 800-325-5052
email: techservice@sial.com

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