Raptor Biphenyl: Fast, Rugged Raptor Columns with Time-Tested Selectivity

With Raptor LC columns, Restek chemists became the first to combine the speed of 2.7 and 5 μm superficially porous particles (also known as SPP or “core-shell” particles) with the resolution of highly selective USLC technology, improving separations and speeding up analysis times with standard HPLC instruments. Raptor then evolved to bring that same improved speed, efficiency, and selectivity to UHPLC analyses by offering 1.8 μm particle columns. Learn more about Raptor LC columns at www.restek.com/raptor

Our top priority when developing our SPP line was to create a version of our innovative Biphenyl. The industry-leading Biphenyl is Restek’s most popular LC stationary phase because it is particularly adept at separating compounds that are hard to resolve or that elute early on C18 and other phenyl chemistries. As a result, the rugged Raptor Biphenyl column is extremely useful for fast separations in bioanalytical testing applications such as drug and metabolite analyses, especially those that require a mass spectrometer (MS). Increasing retention of early-eluting compounds can limit ionization suppression, and the heightened selectivity helps eliminate the need for complex mobile phases that are not well suited for MS detection.

In 2005, Restek was the first to bring you the benefits of the Biphenyl ligand, and we have the experience to maximize the SPP performance of this premier phenyl chemistry for today’s challenging workflows.

Column Description:

Stationary Phase Category: Phenyl (L11) Ligand Type: Biphenyl Particle: 1.8 μm, 2.7 μm, or 5 μm superficially porous particle (SPP or “core-shell” particle) silica Pore Size: 90 Å Surface Area: 125 m2/g (1.8 μm), 130 m2/g (2.7 μm), or 100 m2/g (5 μm)   Column Interaction Profile: Defining Solute Interactions: Polarizability Dispersion   Complementary Solute Interaction: Cation exchange

Recommended Usage: pH Range: 1.5–8.0 Maximum Temperature: 80 °C Maximum Pressure: 1034 bar/15,000 psi* (1.8 μm), 600 bar/8700 psi (2.7 μm); 400 bar/5800 psi (5 μm) * For maximum lifetime, recommended maximum pressure for 1.8 μm particles is 830 bar/12,000 psi. Properties: Increased retention for dipolar, unsaturated, or conjugated solutes. Enhanced selectivity when used with methanolic mobile phase. Ideal for increasing sensitivity and selectivity in LC-MS analyses.   Switch to a Biphenyl when: You observe limited selectivity on a C18. You need to increase retention of hydrophilic aromatics.   Solute Retention Profile: Target Analyte Structures: Aromatic Dipolar   Target Analyte Functionalities: Hydrophilic aromatics Strong dipoles Lewis acids Dipolar, unsaturated, or conjugated compounds Fused-ring compounds with electron withdrawing groups

More Aromatic Selectivity than Ordinary Phenyl-Hexyls

SPP core-shell columns commonly employ traditional phenyl-hexyl stationary phases, but the innovative Biphenyl ligand, developed by Restek’s chemists, is the next generation of phenyl column chemistry. It provides greater aromatic selectivity than commercially available phenyl-hexyl columns [1] and a greater degree of dispersion than conventional phenyls. As a result, the Raptor Biphenyl allows you to more easily separate bioanalytical compounds such as aromatics (Figures 1 and 2), which elute early or are hard to separate on C18 or other phenyl chemistries.

1. In-house testing based on: M.R. Euerby, P. Petersson, W. Campbell, W. Roe, Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns containing phenyl moieties using principal component analysis, J. Chromatogr. A 1154 (2007) 138–151.

	Peaks	tR (min)	Conc. (µg/mL)
1.	Benzene	1.63	500
2.	Nitrobenzene	2.09	100
3.	1,3,5-Trinitrobenzene	4.83	100
4.	2,4-Dinitrotoluene	5.35	100

Column	Raptor Biphenyl (cat.# 9309A55)
Dimensions:	50 mm x 4.6 mm ID
Particle Size:	2.7 µm
Temp.:	40 °C
Standard/Sample
Diluent:	Acetonitrile
Conc.:	100-500 µg/mL
Inj. Vol.:	1 µL
Mobile Phase	Water:Methanol (50:50)
Flow:	1.2 mL/min

Detector	Waters Acquity PDA @ 254 nm
Instrument	Waters ACQUITY UPLC H-Class

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The New Standard for Performance and Durability for SPP Core-Shell LC Columns

Pressure Stability:

One of the greatest advantages of an SPP column is the ability to achieve fast, efficient separations by operating at higher linear velocities than are possible with a conventional fully porous particle column. However, these higher velocities can also result in higher backpressures. Raptor columns were designed to handle the increased pressures needed to achieve Selectivity Accelerated, and handle it far better than other SPP columns on the market (Figure 3).

Reproducibility:

To help keep your productivity high and your lab budget low, we know that Raptor Biphenyl columns must produce exceptional selectivity and fast analysis times not just once, but every time. Ruggedness and repeatability are essential, which is why, from the silica and the bonding technique to the packing process and upgraded hardware, every decision that went into creating this column was made to ensure superlative reproducibility, from injection to injection (Figure 4) and from lot to lot (Figure 5), regardless of column dimension (Figure 6). We also adopted new quality control (QC) specifications to guarantee the retention time stability you need for worry-free MRM analyses.

	Peaks
1.	Cortisol
2.	11-Deoxycortisol
3.	Estradiol
4.	Boldenone
5.	Testosterone
6.	Androstenedione
7.	Progesterone

Column	Raptor™ Biphenyl (cat.# 9309A1E)
Dimensions:	100 mm x 3.0 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
Diluent:	Initial mobile phase
Conc.:	50 ng/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid in water
B:	0.1% Formic acid in acetonitrile
	Time (min) Flow (mL/min) %A %B 0.00 0.700 60 40 3.00 0.700 20 80 3.01 0.700 60 40 5.00 0.700 60 40

Detector	Waters Xevo TQ-S
Ion Mode:	ESI+
Instrument	Waters

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	Peaks
1.	Cortisol
2.	11-Deoxycortisol
3.	Estradiol
4.	Boldenone
5.	Testosterone
6.	Androstenedione
7.	Progesterone

Column	Raptor™ Biphenyl (cat.# 9309A1E)
Dimensions:	100 mm x 3.0 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
Diluent:	Initial mobile phase
Conc.:	50 ng/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid in water
B:	0.1% Formic acid in acetonitrile
	Time (min) Flow (mL/min) %A %B 0.00 0.700 60 40 3.00 0.700 20 80 3.01 0.700 60 40 5.00 0.700 60 40

Detector	Waters Xevo TQ-S
Ion Mode:	ESI+
Instrument	Waters

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Clinically Proven to Optimize Your Bioanalytical Workflows

For over a decade, the Restek Biphenyl has been the column of choice for clinical testing because of its ability to provide highly retentive, selective, and rugged reversed-phase separations of drugs and metabolites. By bringing the speed of SPP to the Biphenyl family, the Raptor Biphenyl provides clinical labs with an even faster option for a wide variety of clinical assays.

Rugged Pain Panels from Urine in Under 3.5 Minutes

Pain panels can be difficult to optimize and reproduce due to the limited selectivity of C18 and phenyl-hexyl phases, but not on the Raptor Biphenyl. Complete your pain panel analysis with a 5-minute cycle time and complete isobaric resolution using Raptor Biphenyl columns (Figure 7). Popular competitor columns offer tailing peaks, longer run times, and coelutions; the Raptor Biphenyl exhibits the selectivity and performance needed for this critical analysis.

	Peaks	tR (min)	Precursor ion	Product ion 1	Product ion 2
1.	Morphine*	1.34	286.2	152.3	165.3
2.	Oxymorphone	1.40	302.1	227.3	198.2
3.	Hydromorphone*	1.52	286.1	185.3	128.2
4.	Amphetamine	1.62	136.0	91.3	119.2
5.	Methamphetamine	1.84	150.0	91.2	119.3
6.	Codeine*	1.91	300.2	165.4	153.2
7.	Oxycodone	2.02	316.1	241.3	256.4
8.	Hydrocodone*	2.06	300.1	199.3	128.3
9.	Norbuprenorphine	2.59	414.1	83.4	101.0

	Peaks	tR (min)	Precursor ion	Product ion 1	Product ion 2
10.	Meprobamate	2.61	219.0	158.4	97.2
11.	Fentanyl	2.70	337.2	188.4	105.2
12.	Buprenorphine	2.70	468.3	396.4	414.5
13.	Flurazepam	2.73	388.2	315.2	288.3
14.	Sufentanil	2.77	387.2	238.5	111.3
15.	Methadone	2.86	310.2	265.3	105.3
16.	Carisoprodol	2.87	261.2	176.3	158.1
17.	Lorazepam	3.03	321.0	275.4	303.1
18.	Diazepam	3.31	285.1	193.2	153.9

*An extracted ion chromatogram (XIC) of the isobars is presented in the inset.

Column	Raptor Biphenyl (cat.# 9309A5E)
Dimensions:	50 mm x 3.0 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
Diluent:	Urine:mobile phase A:mobile phase B (17:76:7)
Conc.:	10-100 ng/mL
Inj. Vol.:	10 µL
Mobile Phase
A:	Water + 0.1% formic acid
B:	Methanol + 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.6 90 10 1.50 0.6 55 45 2.50 0.6 0 100 3.70 0.6 0 100 3.71 0.6 90 10 5.00 0.6 90 10

Detector	AB SCIEX API 4000 MS/MS
Ion Source:	TurboIonSpray®
Ion Mode:	ESI+
Instrument	API LC-MS/MS
Notes	Lorazepam was prepared at 100 ng/mL; all other analytes are 10 ng/mL.

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Column	Competitor SPP Phenyl-Hexyl
Dimensions:	50 mm x 4.6 mm ID
Particle Size:	2.6 µm
Pore Size:	100 Å
Temp.:	22 °C
Standard/Sample
Diluent:	Urine:mobile phase A:mobile phase B (17:76:7)
Conc.:	10-100 ng/mL
Inj. Vol.:	10 µL
Mobile Phase
A:	Water + 10 mM ammonium formate
B:	Methanol + 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.6 95 5 4.00 0.6 0 100 5.00 0.6 0 100 5.10 0.6 95 5 7.00 0.6 95 5

Detector	AB SCIEX API 4000™ MS/MS
Ion Source:	TurboIonSpray®
Ion Mode:	ESI+
Instrument	API LC-MS/MS
Notes	Lorazepam was prepared at 100 ng/mL; all other analytes are 10 ng/mL. Column and conditions used were specifically recommended or published by the manufacturer for this assay.

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Column	Competitor SPP C18
Dimensions:	50 mm x 3.0 mm ID
Particle Size:	2.6 µm
Pore Size:	100 Å
Temp.:	25 °C
Standard/Sample
Diluent:	Urine:mobile phase A:mobile phase B (17:76:7)
Conc.:	10-100 ng/mL
Inj. Vol.:	10 µL
Mobile Phase
A:	Water + 10 mM ammonium formate
B:	Methanol + 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 95 5 3.00 0.5 0 100 4.00 0.5 0 100 4.10 0.5 95 5 5.00 0.5 95 5

Detector	AB SCIEX API 4000™ MS/MS
Ion Source:	TurboIonSpray®
Ion Mode:	ESI+
Instrument	API LC-MS/MS
Notes	Lorazepam was prepared at 100 ng/mL; all other analytes are 10 ng/mL. Column and conditions used were specifically recommended or published by the manufacturer for this assay.

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Bisphenols Resolved Quickly with Biphenyl Selectivity, Not Mobile Phase Adjustments

With health and safety analyses—like the evaluation of bisphenols in consumer products—speed, reliability, and simplicity are crucial. The Raptor Biphenyl column is able to resolve challenging bioanalytical compound sets quickly (e.g., 15 bisphenols in 8 minutes shown below [Figures 8 & 9]) using a simple gradient of mobile phases that require NO additives. When you need baseline resolution for UV detection or high-throughput mass spectrometer applications, especially when fast, trusted, and easy solutions are so vital, the rugged and reproducible Raptor Biphenyl column is the ideal choice.

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
1.	Bisphenol S	0.84	5.00	249.2	108.1	92.1
2.	Bisphenol F	1.62	350	199.3	93.1	105.1
3.	Bisphenol E	2.06	100	213.3	198.3	197.4
4.	Bisphenol A	2.50	100	227.3	212.3	133.1
5.	Bisphenol AF	2.71	2.00	335.2	265.3	177.3
6.	Bisphenol B	3.13	100	241.3	212.4	211.3
7.	Bisphenol C	3.43	350	255.3	240.4	147.3

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
8.	Bisphenol AP	3.98	25.0	289.3	274.3	273.3
9.	Bisphenol Z	4.25	250	267.2	173.4	145.2
10.	Bisphenol G	4.72	250	311.2	295.4	296.4
11.	Bisphenol FL	4.90	50.0	348.8	256.2	-
12.	Bisphenol BP	5.14	50.0	351.2	273.3	274.3
13.	Bisphenol M	5.39	15.0	345.2	330.3	251.4
14.	Bisphenol P	5.67	50.0	345.2	330.4	315.3
15.	Bisphenol PH	6.11	350	379.2	209.4	364.4

Column	Raptor Biphenyl (cat.# 9309252)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	1.8 µm
Pore Size:	90 Å
Temp.:	25 °C
Standard/Sample
Diluent:	75:25 Water:methanol
Conc.:	2.00-350 ng/mL
Inj. Vol.:	2 µL
Mobile Phase
A:	Water
B:	Methanol
	Time (min) Flow (mL/min) %A %B 0.00 0.45 50 50 6.50 0.45 10 90 6.51 0.45 50 50 8.00 0.45 50 50

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC

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	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
1.	Bisphenol S	0.84	5.00	249.2	108.1	92.1
2.	Bisphenol F	1.62	350	199.3	93.1	105.1
3.	Bisphenol E	2.06	100	213.3	198.3	197.4
4.	Bisphenol A	2.50	100	227.3	212.3	133.1
5.	Bisphenol AF	2.71	2.00	335.2	265.3	177.3
6.	Bisphenol B	3.13	100	241.3	212.4	211.3
7.	Bisphenol C	3.43	350	255.3	240.4	147.3

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Product Ion
8.	Bisphenol AP	3.98	25.0	289.3	274.3	273.3
9.	Bisphenol Z	4.25	250	267.2	173.4	145.2
10.	Bisphenol G	4.72	250	311.2	295.4	296.4
11.	Bisphenol FL	4.90	50.0	348.8	256.2	-
12.	Bisphenol BP	5.14	50.0	351.2	273.3	274.3
13.	Bisphenol M	5.39	15.0	345.2	330.3	251.4
14.	Bisphenol P	5.67	50.0	345.2	330.4	315.3
15.	Bisphenol PH	6.11	350	379.2	209.4	364.4

Column	Raptor Biphenyl (cat.# 9309252)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	1.8 µm
Pore Size:	90 Å
Temp.:	25 °C
Standard/Sample
Diluent:	75:25 Water:methanol
Conc.:	2.00-350 ng/mL
Inj. Vol.:	2 µL
Mobile Phase
A:	Water
B:	Methanol
	Time (min) Flow (mL/min) %A %B 0.00 0.45 50 50 6.50 0.45 10 90 6.51 0.45 50 50 8.00 0.45 50 50

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	UHPLC

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Fast Analysis of Sulfur Antibiotics without Coelutions

Even with high-efficiency UHPLC particles, C18 and ordinary phenyl columns fail to achieve baseline separation of sulfonamides. Not only does the Raptor Biphenyl have the selectivity to easily and completely separate these difficult compounds (Figure 10), it does so in well under 5 minutes!

	Peaks	tR (min)
1.	Sulfanilamide	0.41
2.	Sulfadiazine	1.59
3.	Sulfapyridine	1.84
4.	Sulfathiazole	1.96
5.	Sulfamerazine	2.14

	Peaks	tR (min)
6.	Sulfamethazine	2.71
7.	Sulfachlorpyridazine	3.29
8.	Sulfadoxine	3.44
9.	Sulfisoxazole	3.58
10.	Sulfadimethoxine	3.98
11.	Sulfaquinoxaline	4.08

Column	Raptor Biphenyl (cat.# 9309A5E)
Dimensions:	50 mm x 3.0 mm ID
Particle Size:	2.7 µm
Temp.:	20 °C
Standard/Sample
Diluent:	0.1% Formic acid in water
Conc.:	50 µg/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid in water
B:	0.1% Formic acid in acetonitrile
	Time (min) Flow (mL/min) %A %B 0.00 1.5 95 5 2.00 1.5 90 10 4.50 1.5 65 35 4.51 1.5 95 5 5.50 1.5 95 5

Detector	Waters Acquity PDA @ 260, 4.8 nm
Instrument	Waters ACQUITY UPLC H-Class

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	Peaks
1.	Sulfanilamide
2.	Sulfadiazine
3.	Sulfapyridine
4.	Sulfathiazole
5.	Sulfamerazine

	Peaks
6.	Sulfamethazine
7.	Sulfachlorpyridazine
8.	Sulfadoxine
9.	Sulfisoxazole
10.	Sulfadimethoxine
11.	Sulfaquinoxaline

Column	Competitor SPP C18
Dimensions:	50 mm x 3.0 mm ID
Particle Size:	2.6 µm
Pore Size:	100 Å
Temp.:	20 °C
Standard/Sample
Diluent:	0.1% Formic acid in water
Conc.:	50 µg/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid in water
B:	0.1% Formic acid in acetonitrile
	Time (min) Flow (mL/min) %A %B 0.00 1.5 95 5 2.00 1.5 90 10 4.50 1.5 65 35 4.51 1.5 95 5 5.50 1.5 95 5

Detector	Waters Acquity® PDA @ 260, 4.8 nm
Instrument	Waters ACQUITY UPLC H-Class

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Column	Competitor Phenyl-Hexyl
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	1.7 µm
Temp.:	20 °C
Standard/Sample
Diluent:	0.1% Formic acid in water
Conc.:	50 µg/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	0.1% Formic acid in water
B:	0.1% Formic acid in acetonitrile
	Time (min) Flow (mL/min) %A %B 0.00 0.75 95 5 2.00 0.75 90 10 4.50 0.75 65 35 4.51 0.75 95 5 5.50 0.75 95 5

Detector	Waters Acquity® PDA @ 260, 4.8 nm
Instrument	Waters ACQUITY UPLC H-Class
Notes	Flow rate scaled to particle size.

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Experience Selectivity Accelerated. Order the Raptor Biphenyl LC Column today.

Want even better performance when analyzing mycotoxins and other metal-sensitive compounds? Learn more at www.restek.com/inert