TG 01-03

BETASIL® Columns

Introduction
Specifications
100Å high purity silica Phase Particle size Carbon Load Pore Size Endcapping Silica type

High surface area and phase BETASIL C18 3, 5 and 10µm 20% 100Å Yes High purity,
base deactivated
loading BETASIL C8 3, 5 and 10µm 12% 100Å Yes High purity,
Designed for small molecules base deactivated
BETASIL C6 3 and 5µm 11% 100Å Yes High purity,
Type B silica gives good results base deactivated
with acids or bases BETASIL C1 3 and 5µm 4% 100Å Yes High purity,
base deactivated
High phase loading means mo-
BETASIL Phenyl 5µm 11% 100Å Yes High purity,
bile phases require more or- base deactivated
ganic solvent (popular for mass BETASIL Phenyl/Hexyl 3 and 5µm 11% 100Å Yes High purity,
spec) base deactivated
BETASIL Cyano 5µm 7% 100Å Yes High purity,
All reversed phases are end- base deactivated
capped BETASIL Diol 100 5µm 6% 100Å Yes High purity,
base deactivated
Dense bonding results in excel-
BETASIL Silica 100 5µm N/A 100Å N/A High purity,
lent reproducibility and stability base deactivated
Excellent performance with ac-
ids, bases and neutral com-
pounds
Figure 1
Increased Hydrophobic Retention of Neutral Molecules
BETASIL Chromatographic Charac- Sample:
terization 1. Uracil 5. Butyl Benzene
2. Benzene 6. Pentyl Benzene
The BETASIL C18 phase is a high 3. Ethyl Benzene 7. Hexyl Benzene
4. Propyl Benzene 8. Heptyl Benzene
carbon load packing that offers up to
twice the retention of other C18 BETASIL C18 BetaBasic 18 Hypersil BDS C18
packings with lower percent carbon 20%C 13%C 11%C
loading. Figure 1 shows the increased
retention of the BETASIL C18 column 1
1 3 1
when compared to other Thermo
Hypersil-Keystone columns. The in-
creased retention associated with the 4
2
BETASIL column is directly attributed to 2 2
the increased surface area of the 5 3

particles, and higher carbon loading 6

4
5
3
than both Hypersil ® BDS C18 and 7 4 6
5
BetaBasic® 18 columns. 7
6 8
Where longer retention is required or 7
8
701-093 715-173
there is a requirement to use a higher 046-042
percentage of organic solvent in the 0 5 10 15 20 MIN 0 5 10 15 MIN 0 5 10 15 MIN
mobile phase, e.g. to increase LC-MS Columns: 5µm, 150x4.6mm
sensitivity, the BETASIL C18 packing Eluent: 75% ACN / 25% H2O
Flow: 1.25 mL/min
offers an excellent choice. Detector: UV @ 254

1
Stability at Low pH Figure 2
All surface bonded chemistries belong- BETASIL Phase Stability At Low pH
ing to the BETASIL® family are highly
stable under chemical attack. Hydroly- 0.1% TFA gradients at 50°C, pH 1.8
sis of the silane ligand can occur under
acidic conditions, resulting in a loss of
retention and/or selectivity. The effect
becomes more severe as ligand chain
length is reduced, as is observed for the
BETASIL C1 phase. Wettable stationary
phases such as the BETASIL Cyano
packing can also show slightly less re-
sistance to chemical attack, and con-
sequently are recommended only for
use between pH 2 and 8.
In general, BETASIL packings show su-
perior stability to many other packings.
Figure 2 shows BETASIL phases sub-
jected to 25,000 column volumes of ag-
gressive mobile phase at pH 1.8 and
50°C. The BETASIL C18 phase exhibits
virtually no change in retention for the
analysis of a sensitive drug mixture.

Base Deactivated for Better Peak

Shape with Basic Compounds
All BETASIL packings are base deacti-
vated and show excellent peak shape
for basic compounds. Figure 3 shows Figure 3 Figure 4
the analysis of a procainamide test mix-
ture, a mixture that would typically have Base Deactivation of BETASIL C6 Phase Collapse
given rise to broad tailing peaks for the Sample:
1. Uracil
more basic compounds on traditional 2. Procainamide
C18 silicas prepared on a Type A silica. 3. N-Acetyl Procainamide
4. Caffeine
In terms of silanol activity, the BETASIL 2
5. N-Propionyl Procainamide
packing is similar to other Type B silica-
based phases such as BetaBasic® and
1 4 5
BetaMax® Neutral phases.
3

Phase Collapse Phenomenon

Mobile phases that contain a high pro-
portion of water often are employed to
retain highly polar compounds when
using RP-HPLC. Many C18 columns will
show a reversible loss of retention when
exposed to highly aqueous mobile
phase. The rate and degree of reten-
tion loss can vary greatly among differ- 703-002
ent columns. A common explanation
for this retention loss is that the hydro- 0 5 10 MIN

phobic alkyl chains of the stationary BETASIL C6, 5µm, 150x4.6mm

phase are not wettable and appear to Eluent: 10% ACN / 90% 0.05M Phosphate buffer, pH
3.5 with H3PO4
“fold” down on the silica surface to Flow: 1.25 mL/min
Detector: UV @ 254
avoid a highly aqueous, hydrophilic
mobile phase (Figure 4).

2
Regeneration After Phase Collapse Figure 5
In this folded or collapsed state, the High Carbon Load C18 in 100% Aqueous Mobile Phase
alkyl chains are much less able to inter-
act with solutes, resulting in a loss of Sample:
retention. We use the term “chain fold- 1. Tartaric acid
2. Malonic acid
ing” to refer to this reversible loss of re- 4 1,2,3,4 3. Fumaric acid 4
4. Succinic acid
tention induced by highly aqueous mo-
bile phases, although the exact mecha-
nism of reversible retention loss is not
proven. 3 3

The BETASIL® phase is a high carbon

load, high density C18 and conse- 2 1

quently shows the characteristics as-

sociated with chain folding, i.e. loss in 2
retention. However, the situation is com- 1
pletely reversible and the column can
be regenerated simply by flushing with
40% ACN / 60% H2O for 10 minutes bet18001 bet18004 bet18005

(Figure 5).
0 2 4 6 8 MIN 0 2 4 6 8 MIN 0 2 4 6 8 MIN
The phenomena of chain folding can be Partially Folded Completely Folded Regenerated (40% ACN /
almost completely overcome by includ- after >2 hours use 60% H2O for 10 minutes)
ing trace amounts of organic solvent in
BETASIL C18, 150x4.6mm
the mobile phase. Figure 6 shows how Eluent: 0.05M KH2PO 4 + 0.03M H3PO4
just 0.3% n-propanol has been used to Flow: 1.0 mL/min
Detector: UV @ 210
retard the loss in retention associated
with chain collapse. Further information
on chain collapse can be found in Tech-
nical Bulletin TB99-01.
Figure 6 Figure 7
Trace Organics in Mobile Phase
Choices in Stationary Phase Retard Chain Folding Polyphenols
Chemistries
Sample:
BETASIL columns are available in a wide 1. Tartaric acid
Sample:
2. Malonic acid
range of reversed phase packings, in- 3. Fumaric acid
1. Pyrogallol
4 2. Hydroquinone
cluding C18, C8, C6, Phenyl, Cyano, 4. Succinic acid
3. Resorcinol
and Phenyl/Hexyl. The BETASIL Phe- 4. Phenol

nyl/Hexyl phase provides a combination 2 3 Initial 2

of straight-chain C6 groups and phenyl 1
groups, resulting in a mixed-mode
separation. The C6 chain exhibits clas-
bet18016
sical reversed phase retention, while the
phenyl ring provides special selectivity 4
for polar groups (Fig. 7).
2 3

BETASIL columns also offer choices for 3

After 8 hours
normal phase chromatography, includ- 1

ing silica and diol phases in 4 different 1

pore sizes from 60Å to 300Å, and cy- bet18020
4

ano. Diol phases provide higher polar H730-007

selectivity than cyano with less water 0 2 4 6 MIN
0 5 10 15 20 25 30 35 MIN
sensitivity than bare silica.
BETASIL C18, 150x4.6mm BETASIL Phenyl/Hexyl, 5µm, 150x4.6mm
Eluent: 0.05M KH2PO 4 + 0.03M H3PO4 + 0.3% n-propanol Eluent: 5mM Acetic Acid
Flow: 1.0 mL/min Flow: 1.0 mL/min
Detector: UV @ 210 Detector: UV @ 280
Temp.: 25°C

3
 Carbamate/Urea
Organic Acids Benzodiazepines Pesticides & Herbicides
Sample: Sample: Sample:
1. Ascorbic Acid 1. Nitrazepam 1. Tebuthiuron
2. Acetaminophen 2. Oxazepam 2. Carbofuran
3. Benzoic Acid 3. Temazepam 3. Carbaryl
4. Salicylic Acid 4. Nordiazepam 4. Diuron
5. Diazepam 5. Barban
6. Medazepam

2 4

4
3
5
2

6
1
1

2
3 H730-008 H730-005
4 H730-006

0 2 4 6 8 10 12 14 16 18 MIN 0 2 4 6 8 MIN 0 2 4 6 8 10 12 MIN

BETASIL® Phenyl/Hexyl, 5µm, 150x4.6mm BETASIL Phenyl/Hexyl, 5µm, 150x4.6mm BETASIL Phenyl/Hexyl, 5µm, 150x4.6mm
Eluent: 80% 20mM KH2PO4, pH 2 / 20% MeOH Gradient: A: H2O Gradient: A: H2O
Flow: 1.0 mL/min B: MeOH B: ACN
Detector: UV @ 254 Time %B Time %B
Temp.: 25°C 0 15 0 15
7 85 7 85
Flow: 1.0 mL/min Flow: 1.5 mL/min
Detector: UV @ 235 Detector: UV @ 254
Temp.: 25°C Temp.: 25°C

Procainamides Cyclosporin Assay

1
500 ng / mL Real Sample:
1
Sample:
standard 383 ng / mL
1. Uracil
2. Procainamide 1
3. N-Acetyl Procainamide
4. Caffeine
5. Propionyl Procainamide Sample: 2
2
1. Cyclosporin
2 2. Internal Standard

c1-std c1-pat

708-002
0 5 MIN 0 5 MIN
0 4 8 MIN
BETASIL C6, 5µm, 150x4.6mm BETASIL C1, 5µm, 150x4.6mm
Eluent: 10% ACN / 90% 0.05M KH2PO4, Eluent: 35% ACN / 65% 0.05M KH2PO4, pH 7
pH 3.5 with H3PO4 Flow: 1.25 mL/min
Flow: 1.25 mL/min Detector: UV @ 254
Detector: UV@ 254

4
 Common Analgesic Water-Soluble Vitamins Pesticides

Sample: Sample: Sample:

1. Acetaminophen 1. Pyridoxine 1. Dimethoate 5. Ethyl Parathion (DNTP)
2. Caffeine 2. Thiamine 2. Methyl Parathion 6. Diazinon
3. Acetylsalicylic Acid 3. Niacinamide 3. Malathion 7. EPN
4. Benzoic Acid 4. Cyanocobalamin 4. Fenitrothion
5. Riboflavin

3
1 4
1

4 7
3 4 5 5
2 2

3 6

701-068 vit10050
beta0031
0 2 MIN 0 5 10 MIN 0 5 10 15 20 25 MIN

® TM
BETASIL C18 PIONEER , 5µm, 50x3mm BETASIL C18, 5µm, 150x4.6mm BETASIL C18, 5µm, 150x4.6mm
Eluent: A: 0.05M Ammonium Acetate, pH 4 Gradient: A: 0.05M K2HPO4, pH 6.5 Gradient: 50% ACN / 50% H2O, step to
B: 70% ACN% / 30% MeOH B: ACN 60% ACN / 40% H2O at 12 min.
85:15 A:B 2% B to 20% B in 15 min Flow: 1.0 mL/min
Flow: 2.5 mL/min Flow: 1.0 mL/min Detector: UV @ 235
Detector: UV@ 254 Detector: UV @ 265

Steroids by Normal Phase

Sample:
1. Progesterone 7. 11α-Ketoprogesterone
2. 20α-Hydroxy-4-pregnene-3-one 8. Corticosterone
3. 17α-Hydroxyprogesterone 9. Cortisone Acetate
4. Deoxycorticosterone 10. Hydrocortisone
5. 11α-Hydroxyprogesterone Acetate 11. Cortisone 1 3
6. 11α-Hydroxyprogesterone

1
6
3
4,7 6

1 2 5
2 4,7
2
3 5 8
4,7 6
9 10
10
5 8

9 9
8
11
10 11

11

725-001 726-001 728-001

0 5 10 15 MIN 0 5 10 MIN 0 5 MIN

BETASIL Diol 60, 5µm, 150x4.6mm BETASIL Diol 100, 5µm, 150x4.6mm BETASIL Diol 300, 5µm, 150x4.6mm
Eluent: 85% Isooctane / 14.7% EtOH / 0.3% H2O Eluent: 85% Isooctane / 14.7% EtOH / 0.3% H2O Eluent: 85% Isooctane / 14.7%
Flow: 1.2 mL/min Flow: 1.2 mL/min EtOH / 0.3% H2O
Detector: UV @ 254 Detector: UV @ 254 Flow: 1.2 mL/min
Detector: UV @ 254

BetaBasic, BetaMax, BETASIL, Hypersil, and HyPURITY are registered Trademarks of Thermo Hypersil-Keystone.
PIONEER is a Trademark of Thermo Hypersil-Keystone.
©2002 Thermo Hypersil-Keystone. All Rights Reserved.