BioBasic SCX and AX Columns - Chromatopak.com

BioBasic ® SCX and AX 

Columns 

TG02-01 

Silica-based ion exchange columns for 

cation and anion exchange chromatography

2 

Technical Guide 

BioBasic ® SCX and AX Columnss 

Using BioBasic ® SCX and AX for 

Ion Exchange Chromatography 

BioBasic ion exchange columns are the first in a new generation of HPLC columns that make use of polymeric ion 

exchange ligand technology bound to a high quality base deactivated silica. This unique combination gives rise to a 

highly efficient and stable polymeric coating that is very robust and reproducible. The columns can be used in any one 

of several modes of operation: ion exchange, normal phase, and hydrophilic interaction chromatography. 

BioB BioB BioBaaaaasssssic BioB BioB ic ic ic ic AX AX AX AX AX fffor 

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or or or Anion Anion Anion Anion Anion Ex Ex Exccccchhhhhan Ex Ex an an anggggge an e e e e Chr Chr Chrom Chr Chrom 

om omat omat 

at atogr atogr 

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aph aph aph Anion exchange chromatography is an electrostatic interaction 

between a negatively charged analyte and a positively charged stationary phase. Retained analytes are eluted 

by the introduction of a salt that competes with the analyte for the ion exchange site. 

BioB BioB BioBaaaaasssssic BioB BioB ic ic ic ic SCX SCX SCX SCX SCX fffor 

ffor 

or or or CC 

Cation CC 

ation ation ation ation Ex Ex Exccccchhhhhan Ex Ex an an anggggge an e e e e Chr Chr Chrom Chr Chrom 

om omat omat 


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aph aphyyyyy..... aph Cation exchange chromatography is an electrostatic interaction 

between a positively charged analyte and a negatively charged stationary phase. Retained analytes are 

eluted by the introduction of a salt that competes with the analyte for the ion exchange site. 


ffor 

or or or Norm Norm Normal Norm Normal 

al al al Ph Ph Phaaaaase Ph Ph se se se se Chr Chr Chrom Chr Chrom 

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aph aphyyyyy. aph Normal phase chromatography is governed by the ability of the 

analyte to hydrogen bond with the stationary phase. Retention is controlled by adding small quantities of a 

competing solvent with hydrogen bonding properties to the mobile phase (e.g. ethyl acetate). 


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aph aphyyyyy aph (HILIC). (HILIC). (HILIC). (HILIC). (HILIC). HILIC is similar to normal phase chromatography. 

In this mode however, portions of the mobile phase are aqueous. In the HILIC mode, retention increases 

with the polarity of the analyte and retention decreases with the polarity of the eluent. The stationary phase 

easily adsorbs water from the mobile phase making it very hydrophilic. 

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ionic ionic s sstr 

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Ap Ap Apppppplic Ap Ap lic lic lication lication 

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� Nuc Nucleotide 

Nuc eotide eotides eotide 

� Pr Prot Pr ot otein ot ein eins ein and and and peptide peptides peptide 

� Ar Arom Ar om omatic om atic ac acid ac id ids id 

� Sug ug ugar ug ar ars ar 

� Pho Phospho Pho pho pholipid pho lipid lipids lipid 

� Antib Antibiotic Antib iotic iotics 

iotic 

Figure 1a shows the separation of 12 nucleotides using BioBasic AX in 

the ion exchange mode. 

Figure 1b shows the separation of 5 proteins using the BioBasic SCX 

column in ion exchange mode. 

figure 1a. Nucleotides 

2 

1 3 

Sample: 1. CMP 

2. UMP 

3. AMP 

4. GMP 

5. CDP 

6. ADP 

4 

5 

6 

7 

8 

7. UDP 

8. GDP 

9. CTP 

10. ATP 

11. UTP 

12. GTP 

9 10 11 12 

731-001 

0 5 10 15 20 25 30 35 MIN 

BioBasic AX, 5µm, 150x4.6mm 

Gradient: A: 5mM KH PO , pH 3.2 

2 4 

B: 0.75M KH PO , pH 3.2 

2 4 

0-100% B in 30 min 

Flow: 1.0 mL/min 

Detector: UV @ 254 

figure 1b. Proteins with Tris Buffer 

Sample: 

1. Trypsinogen 

2. Ribonuclease A 

3. Chymotrypsinogen A 

4. Cytochrome C 

5. Lysozyme 

1 

BioBasic SCX, 5µm, 150x4.6mm 

Gradient: A: 0.02M Tris, pH 6 

B: A + 1.0M Na Acetate, pH 6 

0-100% B in 60 min 



2 

3 

4 

5 

732-002 

0 5 10 15 20 25 30 35 MIN

BioBasic AX Polyethyleneimine Structure and Ion Exchange Capacity 

1. BioBasic AX is an anion exchange packing consisting of polyethyleneimine (PEI) covalently bound to a highly 

base deactivated 300Å, 5µm silica. Polyethyleneimine has a polymeric structure (Figure 2). 

Particle size 5µm 

Pore size 300Å 

Ion exchange ligand PEI 

Ion exchange capacity 0.22mequivalents/gram 

Surface chemistry schematic see Figure 2 

Figure 2. Structure of PEI 

Branching during polymerization leads to 30% primary, 40% secondary, and 30% tertiary amines. 

The ion exchange capacity of BioBasic AX is measured by titration. Details concerning the method and calculation 

are presented in Figure 3b. 

The measured capacity is 0.22 mequivalents/gram. This measured capacity compares to other low capacity 

ion exchangers available. The low capacity allows the use of moderate to low buffer concentrations when 

eluting samples from the column – a real benefit where mass spectrometry(MS) is the mode of detection. 

BioBasic AX was primarily developed as a high performance ion exchange packing, but it has also been 

successfully used for HILIC and Normal Phase Chromatography (NPC). The ion exchange mode and HILIC 

modes are discussed more completely in the following paragraphs. 

BioBasic SCX Structure and Ion Exchange Capacity 

2. BioBasic SCX packing is a strong cation exchange packing consisting of highly base deactivated 300Å, 5µm 

silica with a sulphonic acid derivatized hydrophilic polymer coating that provides excellent stability and an 

excellent surface for protein chromatography i.e. relatively minor secondary interactions (Figure 3a). 

Particle size 5µm 

Pore size 300Å 

Ion exchange ligand Sulphonic acid 

Ion exchange capacity 0.07 mequivalents/gram 

Surface chemistry schematic see Figure 3a 

Figure 3a. Sulfonic Acid Structure 

The ion exchange capacity of BioBasic SCX is measured by titration. Details concerning the method and 

calculation are presented in Figure 3b. 

The measured capacity is 0.07 mequivalents/gram. This capacity is actually slightly less than other low 

capacity ion exchangers available. While this lower capacity means that sample concentration may need to be 

reduced so as not to overload the column, it does allow for the use of lower buffer concentrations for elution 

than typically expected. This characteristic is quite beneficial for the MS mode of detection. 

BioBasic SCX was primarily developed as a high performance ion exchange packing for traditional analytical 

separations, but it has also been shown to be particularly proficient at fractionation of complex mixtures of 

proteins. 

3

4 


BioBasic ® SCX and AX Columns 

Figure 3b. BioBasic ® AX Ion Exchange Capacity Determination by Titration 

Basic Principles of Ion Exchange Chromatography 

Purpose: To determine the fundamental 

ion exchange capacity and 

demonstrate the effective weak 

anion exchange pH range for 

Biobasic AX. 

Reference: A.J. Alpert and F. E. Regnier J. 

Chrom 185 (1979) 

pgs 375-392 

Method: 0.600g of lot R0J17 was sus 

pended in 20mL of 0.5M 

sodium chloride. The stirred 

suspension was then titrated 

using 0.0408N HCL. The pH 

was monitored using a pH meter 

and probe rather than a visual 

indicator. 

Most ion exchange packings fall into two groups: those which contain acid groups, such as sulphonic acid or carboxylic 

acid, for the separation of cationic compounds, and those which contain a basic group, such as an amine or quaternary 

amine, for the separation of anionic compounds. It is important to buffer the mobile phase in this mode of chromatography 

to control the ionization of both the analyte and the packing, since the ionic state of both affects the acid-base 

equilibria between analyte and ion exchange packing. A strong anion exchange media such as BioBasic AX will be ionized 

over most of the pH range 2-8. A weak anion exchange media can be turned “on” and “off” within that range through 

changes in the pH of the mobile phase. It is important to emphasize that, while the ion exchange capacity of BioBasic AX 

will be maximized at low pH, it will still have some ionic character and function as an ion exchanger even at pH 8. This 

makes BioBasic AX very versatile and makes pH a powerful tool for modifying selectivity. 

The anion exchange process may be represented by the equilibrium [1], 

which would apply for an anion exchange material such as BioBasic AX : 

where C- is the counter ion to the fixed ion exchange site, ~NH + , present in the eluent. Analyte X- can then displace C- to 

give the ion pair NH + X- . For effective chromatography, this displacement reaction must be at equilibrium. Retention is 

therefore controlled by the equilibrium [1] for which the equilibrium constant KIE is given by equation [2]: 

The capacity factor, k, of an analyte (X - ) is a more useful chromatographic paramater and is proportional to the distribution 

coefficient, D IE . Its relationship equilibrium constant, K IE , is given by equation [3]: 

Since the concentration of the ion exchange sites, [~NH + X - ], is constant and fixed by the rigid structure of the matrix, k is 

inversely proportional to the concentration of the counter ion (ionic strength) in the eluent: 

Desorption is then brought about by increasing the salt concentration as shown by equation [4]. 

[1] 

[2] 

[3] 

[4]

Ion Exchange Chromatographic 

Characteristics of BioBasic AX and 

BioBasic SCX 

In ion exchange chromatography, 

conditions are employed that permit 

the sample components to move 

through the column at different 

speeds. The higher the net charge of 

the analyte, the higher the ionic 

strength needed to bring about 

desorption. At a certain high level of 

ionic strength, all the sample 

components are fully desorbed and 

move down the column with the 

same speed as the mobile phase. 

Conditions that lie somewhere in 

between total adsorption and total 

desorption will provide the optimal 

selectivity for a given pH value of the 

mobile phase. The effect of ionic 

strength on analyte retention is clearly 

shown in Figures 4a and 4b. The term 

adsorption used here in a general 

sense refers to the attraction of an 

analyte ion for the ionic sites of the 

packing. It should not be confused 

with the same term used for 

adsorption chromatography where 

traditional hydrogen bonding is the 

mechanism by which analytes are 

retained at the surface. 

The competition between analyte and 

salt for the ion exchange site is largely 

controlled by the concentration and 

nature of the salt ion. Choice of the 

appropriate counter ion is therefore of 

significant importance in adjusting 

retention. One usually employs H + , 

Na + , K +, , Ca2+ - for cation and NO , 3 

2- 3- SO , PO4 , etc. for anion exchangers. 

4 

For example, by substituting the 

eluent ion of a cation exchanger in the 

series H + , Na + , K + ,Ca2+ the relative 

strength between counter ion and 

fixed ion increases and retention of a 

given cation (analyte) decreases. 

The concentration, or alternatively 

ionic strength, of the counter ion also 

plays an important role in controlling 

retention. At low ionic strengths, all 

components with affinity for the ion 

exchanger are strongly adsorbed at 

the top of the ion exchange column 

and elution is slow. As the ionic 

strength of the mobile phase is 

increased by increasing the concentration 

of a salt, salt ions compete 

more strongly with the analyte for the 

ion exchange site. The sample starts 

moving down the column more 

quickly, causing the elution time to be 

reduced. Increasing the ionic strength 

even more causes a further reduction 

in elution time until the analyte does 

not spend any time in the stationary 

phase. 

Figure 4a. Effect of Buffer Concentration 

4 

1 

3 

2 

5 8 

7 

6 

4 

1 

3 

2 

5 

7 

6 

1 

1 

2 

3 

4 5 

2 3 

6 

4 5 

7 

6 

8 

BioBasic AX 

90mM 

70mM 

8 

7 

Sample: 

1. Uracil 

2. Shikimic Acid 

3. Ascorbic Acid 

4. Phenylacetic Acid 

5. Sorbic Aci 

6. Benzoic Acid 

7. Vanillic Acid 

8. p-Coumeric Acid 

50mM 

731-008 

0 4 8 12 MIN 

8 

30mM 

BioBasic ® AX, 5µm, 50x4.6mm 

Eluent: A: NH 4 Acetate, pH 5.7 

B: Acetonitrile 

95%A / 5%B 



Sample components can vary 

considerably in their adsorption to the 

ion exchange sites so that a single 

value of the ionic strength cannot 

make all analytes (both strongly and 

weakly retained) pass through the 

column in a reasonable time. In such 

cases, a salt gradient is applied. This 

causes a gradual increase in ionic 

strength in the mobile phase. This 

gradient gradually desorbs the sample 

components in the order of increasing 

net charge, so that components are 

picked off one at a time from the 

surface to provide separation of the 

mixture. Thus the salt gradient 

compresses a chromatogram so as to 

elute components with widely 

different adsorptive properties within a 

reasonable time. 

Figure 4b. Effect of Buffer Concentration 

1 

1 

1 

2 

2 

2 

3 

4 

5 6 

3 

5 6 

4 

3 


30mM 

20mM 

Sample: 

1. Caffeine 

2. Pyridine 

3. Diphenhydramine 

4. Dextromethorphan 

5. Pseudoephedrine 

6. Nicotine 

5 6 

4 

10mM 

732-003 

0 2 4 6 MIN 


Eluent: 60% NH 4 Acetate, pH 4.5 / 40% ACN 



5

6 



pH Control 

pH can have considerable effect on 

retention and selectivity. This is because: 

1. a shift in pH that causes the 

analyte to change from its 

ionized state to its neutral 

state prevents the analyte 

from taking part in the ion exchange 

process and consequently 

retention is reduced. 

2. a shift in pH that causes the ion 

exchange site to change from 

its ionized state to a neutral 

state essentially eliminates 

sites available for ion exchange 

and retention is lost. 

Maximizing Column Efficiency and 

Resolution 

High column efficiency (number of theoretical 

plates) is an important factor to 

consider when trying to maximize the 

potential resolution in a given separation. 

In ion exchange chromatography 

the sharpness of a peak is governed 

not only by how well the column is 

packed but also by the mass transfer 

characteristics of the ion exchange 

mechanism itself. The term mass transfer 

used here refers to the efficient migration 

of the analyte from the mobile 

phase on to the stationary phase ion 

exchange site and then back into the 

the mobile phase from the ion exchange 

site. If this process is not very rapid, 

broad peaks can result with a possible 

loss in analyte resolution. Figure 5a illustrates 

how the column efficiency increases 

as the flow rate is reduced for 

a 150 x 4.6mm column. Depending on 

the complexity of the analysis, a tradeoff 

is required between time and efficiency. 

Even at moderate flow rates, 

the column efficiency is still very impressive 

for ion exchange. 

The mass transfer component of the 

Van Deemter equation can often be improved 

through temperature control. 

The mass transfer can be increased by 

increasing the temperature at which the 

separation takes place. The way in which 

column temperature is achieved is also 

of significant importance. Figure 5b 

shows the effect that increasing column 

temperature has on efficiency. It is important 

to note the role of preheating 

the mobile phase on the separation. 

For example, heating only the column 

itself and not heating the solvent supplied 

to it can actually increase band 

spreading and reduce column efficiency 

as shown in Figure 5b. Heating the solvent 

prior to its arrival at the column 

prevents this band spreading and improves 

the mass transfer associated 

with the ion exchange mechanism. The 

result is an increase in the column efficiency 

for any given flow rate. For further 

information on temperature control 

please contact Thermo Hypersil- 

Keystone and request the Hot Pocket 

Product Bulletin. 

Figure 5a. The Effect of Flow Rate 

on Efficiency in Ion Exchange Mode 

Figure 5b. The Effect of Temperature 

on Efficiency in Ion Exchange Mode 

Attributes of BioBasic Ion Exchange Packings 

BioBasic AX and SCX are the first in a new generation of high performance 

ion exchange columns for HPLC. They offer: 

1. Proven batch-to-batch reproducibility 

2. High efficiency, measured at >80,000 plates/meter 

3. Excellent phase stability 

4. Suitability for the separation of small highly polar pharmaceutical 

compounds and also larger biochemical molecules such as proteins 

and peptides 

5. A broad application range 

6. BioBasic AX offers - three modes of retention: a) anion exchange, 

b) normal phase and c) hydrophilic interaction chromatography 

7. BioBasic SCX has proven to be highly proficient at protein digest 

fractionation and is consequently ideal for applications in the field 

of proteomics.

Lot to Lot and Column to Column 

Reproducibility 

Each lot of BioBasic AX and BioBasic 

SCX is bonded to very tight specifications. 

Lots are tested chromatographically 

(Figure 6a and 6b) with ionic 

probes using buffered mobile phase 

conditions in order to fully characterize 

the ionic interactions that take place at 

the packing surface. 

Individual columns packed from any 

given lot are also tested chromatographically 

in order to ensure the high 

performance that might be expected 

from the spherical 5µm base deactivated 

silica particles that are used as 

the base to both the BioBasic AX and 

SCX polymeric bonded phases (Figure 

7). Typical efficiencies are greater than 

80,000 plates per meter when tested 

in the normal phase mode. 

Phase Stability 

The unique way in which the 

hydrophilic polymer is bonded to the 

highly base deactivated silica 

provides a very stable surface on 

which chromatography takes place. 

BioBasic SCX Stability 

Stability trials have been carried out 

at pH 2.5 and pH 6 on BioBasic SCX. 

The trials involved testing two 

separate columns, each subjected 

to: 

a. >14,000 column volumes of 

0.05M KH PO at pH 2.5, 

2 4 

adjusted with H PO 3 4 

b. >15,000 column volumes of 

0.05M KH PO at pH 6, 

2 4 

adjusted with KOH 

The columns were removed at 

regular intervals and tested chromatographically 

using the test 

procedure outlined in Figure 8. The 

retention of Nicotine was then 

recorded and plotted as shown in 

Figure 8. 

figure 6a. BioBasic ® AX 

Batch-to-Batch Reproducibility figure 6b. Peptides 

1 

Sample: 

1. Uracil 

2. UMP 

3. AMP 

4. GMP 

2 

731-009 

0 2 4 6 8 MIN 


Eluent: 0.05M KH 2 PO 4 , pH 4.68 



3 

4 

POK10 

ROJ18 

POK11 

figure 7. Normal Phase Efficiency 

1 

2 

3 

4 

BioBasic AX 

N = 81,628 

Sample: 

1. Toluene 

2. Nitrobenzene 

3. o-Nitroaniline 

4. m-Nitroaniline 

5. p-Nitroaniline 

0 2 4 6 MIN 

Columns: 5µm, 150x4.6mm 

Eluent: 85% IsoOctane / 15% EtOH (0.3% HOH) 



5 

731-011 

BioBasic AX Stability 

Stability trials have been carried out at 

pH 2.5 and pH 8 on BioBasic AX. The 

trials involved testing two separate columns, 

each subjected to: 

Sample: 

1. Ac-G-G-G-L-G-G-A-G-G-L-K-amide 

2. Ac-K-Y-G-L-G-G-A-G-G-L-K-amide 

3. Ac-G-G-A-L-K-A-L-K-G-L-K-amide 

4. Ac-K-Y-A-L-K-A-L-K-G-L-K-amide 

002-145 

0109010A 

732-006 

0 2 4 6 8 10 12 MIN 


Eluent: A: 10mM KH 2 PO 4 in 25% ACN 

(pH adjusted to 4.8 with H 3 PO 4 ) 

B: A + 0.5M NaCl 

0->50% B in 20 min 




N = 82,305 

1 

0 1 2 3 MIN 

2 

3 

002-148 

732-001 

a. a. >15,000 column volumes of 0.05M 

KH PO at pH 2.5, adjusted with 

2 4 

H PO 3 4 

b. >15,000 column volumes of 0.05M 

KH PO at pH 8, adjusted with KOH 

2 4 

7

8 



The columns were removed at regular 

intervals and tested chromatographically 

using the test procedure outlined 

between Figures 9a and 9b. The 

retention of GMP was then recorded 

and plotted in these Figures. 

pH 2.5 Conditions 

Eluent: 60% 10mM NH 4 Acetate, 

pH 4.5 / 40% ACN 



Figures 9a & 9b. Stability of BioBasic 

Column retention restored 

® AX at pH 2.5 and at pH 8 

pH pH 2.5 

2.5 

pH pH 8 

8 

BioBasic SCX, 50x4.6mm 

The results show that at both pH 2.5 

and pH 8 some loss in column 

retention does occur. This is largely 

due to the adsorption of impurities 

from the phosphate buffer itself. 

These are easily removed by using a 

Figure 8. BioBasic SCX Column Stability @ pH 2.5 and pH 6.0 

pH 6 Conditions 

Eluent: 70% 10mM NH4 Formate, 

pH 3.1 / 30% ACN 



pH 8 and pH 2.5 Conditions 


Eluent: 5mM KH PO , pH 6.5 

2 4 



Column retention restored 

high concentration salt gradient 

regeneration program. Once this has 

been done, capacity factors are 

restored to within 5% of their original 

values. This observation was made for 

both pH trials, as shown by the circled 

areas in Figures 9a and 9b. The 

regeneration procedure used is given 

in Figure 10. 

Figure 10. Regeneration of 

Retention with Salt Gradients 

Regeneration 

Procedure: 

Conditions: A: 0.1M KH 2 PO 4 , pH not adjusted 

B: 0.7M KH 2 PO 4 , pH not adjusted 

Gradient: 0-100% B in 15 min. 

- Run several gradients to clean column and 

free ion exchange sites. 

1 

1 

1 

2 

2 

2 

3 

3 

Sample: 

1. Uracil 

2. UMP 

3. CMP 

3 

After 15,000 Column 

Volumes @ 

pH 2.5 0.05M KH 2 PO 4 

4 

New Column 

4 5 

4 

5 

Regeneration After 3 

Strong Salt Gradients 

96% of Original Retention 

731-012 

0 5 10 MIN 

5 

4. AMP 

5. GMP

Tips on how to switch from chromatographic mode to chromatographic mode 

When changing from one mode of chromatography to another, i.e. from buffered ion exchange conditions to 

normal phase/HILIC conditions, performance difficulties can occur. This manifests itself in an apparent loss of 

column efficiency and a loss in integrity of the chromatogram. In order to avoid this difficulty it is strongly 

recommended that the following wash procedure be employed when changing from ion exchange mode to 

normal phase mode (at least five column volumes of each). 

To change from a buffered solvent system to a normal phase solvent system: 

– flush the column with water – flush the column with 1M ammonium acetate 

– flush the column with water – flush the column with ethanol 

– run the column in normal phase solvents, i.e. hexane, ethyl acetate, etc. 

To change from normal phase solvent system to a buffered mobile phase: 

– flush the column with ethanol – flush the column with water 

– flush the column with buffered solvent of choice 

Hydrophilic Interaction Chromatography 

(HILIC) Mode using BioBasic AX 

In the HILIC mode of chromatography, 

the stationary phase must be 

extremely polar. Polar analytes 

passing through the column in 

aqueous/organic mobile phase 

have a high affinity for this surface. 

The equilibrium that exists 

between the analyte in the 

stationary phase and the analyte in 

the mobile phase is controlled by 

the proportion of aqueous solvent 

present in the mobile phase. The 

higher the aqueous content of the 

mobile phase, the stronger the 

affinity the polar analyte has for it 

and the equilibrium moves 

towards that analyte in the mobile 

phase. The analyte then passes 

through the column more quickly, 

i.e retention time is reduced. This 

is the opposite of that seen for 

reversed phase chromatography 

where an increase in aqueous 

content results in an increase in 

retention. HILIC can also be used 

in gradient elution mode. Two 

figure 11a. Sugars in HILIC Mode figure 11b. Phospholipids in HILIC Mode 

1 

2 

3 


Eluent: 75% ACN / 25% H 2 O 


Detector: ELSD 

4 

5 

731-003 

0 2 4 6 MIN 

Sample: 

1. Glucose 

2. Maltose 

3. Maltotriose 

4. Maltotetraose 

5. Maltopentaose 

examples of BioBasic AX using 

this mode are given in Figures 11a 

and 11b. However, in Figure 11b 

there could be some mixed-mode 

interaction taking place. Some ion 

Sample: 

1. L-erythro-Sphingosine 

2. L-a-Phosphatidyl choline, egg 

3. Phosphatidyl ethanolamine, bovine 

4. lyso-Lecithin, egg 

2 

1 

731-013 

0 5 10 15 MIN 

BioBasicAX, 5µm, 150x4.6mm 

Gradient: A: 95% ACN / 5% 5mM NH 4 Formate, pH 6.13 

B: 80% ACN / 20% 5mM NH 4 Formate, pH 6.13 

0%B 0-5 min., 0-100%B 5-15 min. 


Detector: ELSD 

exchange interactions may occur 

because BioBasic AX is partially 

charged and phospholipids may 

have negative charge at this pH. 

3 

4 

9

10 



Aromatic Acids Antibiotics Proteins 

Sample: 

1. Uracil 

2. Shikimic Acid 

3. Ascorbic Acid 

4. Phenylacetic Acid 

1 

2 

3 

Sample: 

1. Cyanocobalamin (B12) 

2. Riboflavin (B2) 

3. Pyridoxine (B6) 

4. Thiamine (B1) 

4 

2 

1 

3 

4 

5 

6 

7 

5. Sorbic Acid 

6. Benzoic Acid 

7. Vanillic Acid 

8. p-Coumeric Acid 

731-002 

732-007 

0 2 4 6 8 10 12 14 16 MIN 


Eluent: A: 50mM KH 2 PO 4 , pH 3.0 

B: 0.5M KH 2 PO 4 , pH 3.0 

Gradient: 10% B for 6 min, 100% B at 6.1 min 



Temp: 50° 

*Note: Baseline subtracted 

1 

1 

2 

3 

Sample: 

1. Ac-G-G-G-L-G-G-A-G-G-L-K-amide 

2. Ac-K-Y-G-L-G-G-A-G-G-L-K-amide 

3. Ac-G-G-A-L-K-A-L-K-G-L-K-amide 

4. Ac-K-Y-A-L-K-A-L-K-G-L-K-amide 

3 

2 

731-004 

0 2 4 6 8 MIN 0 4 8 12 MIN 0 5 10 15 20 25 30 35 40 MIN 


Eluent: A: 70mM NH 4 Acetate, pH 5.7 


95%A / 5%B 



8 

Sample: 

1. Cephaloridine 

2. Amoxicillin 

3. Ampicillin 

4. Cephalosporin C 

5. N-Acetylpenicillamine 

6. Penicillin G 


Eluent: A: 10mM NH 4 Acetate, pH 6 


90%A / 10%B 



Temp: 35°C 

Vitamins Substituted Peptides 

4 

5 

6 

0 2 4 6 8 10 12 14 16 MIN 


Eluent: A: 10mM KH 2 PO 4 in 25% ACN (pH adjusted to 4.8 with H 3 PO 4 ) 

B: A + 0.5M NaCl 

0->50% B in 20 min 



4 

Sample: 

1. b-Lactoglobulin B 

2. b-Lactoglobulin A 


Gradient: A: 0.02M TRIS, pH 6 

B: A + 1M NaAcetate, pH 6 

0-100%B in 40 min. 



1 

2 

731-005 

732-005

BioBasic BioBasic BioBasic BioBasic BioBasic®®®®® 

SCX SCX SCX SCX SCX for for for for for 2D 2D 2D 2D 2D Proteomics 

Proteomics 

Proteomics 

Proteomics 

Proteomics 

Proteomics is the study of proteins 

and their interaction within organisms, 

including the human body. Diseased 

states can frequently be traced to 

problems with protein expression and 

interaction. Proteomics accelerates 

drug discovery, since over 95 percent 

of all pharmaceuticals target proteins. 

Now that the genome has been 

sequenced, accelerated protein 

research is the next logical step, 

assisted by rapid improvements in the 

existing technologies. 

Much of the work in proteomics is 

conducted with electrophoresis and 2- 

D gels. However, the volume of work 

required calls for methods and tools 

that offer greater speed and sensitivity 

to produce rapid breakthroughs. The 

Thermo Finnigan ProteomX Workstation 

is one such tool, combining HPLC 

and tandem MS with ion trap 

technology. 

2D proteomics combines reversed 

phase and ion exchange chromatography 

to increase the efficiency of 

protein identification methods (see 

Figure 12). Co-eluting components 

can be separated into different 

fractions, in effect removing interferences 

from more abundant co-eluting 

species. A typical two-dimensional 

LC/MS method involves separating 

fractions of protein digests by cation 

exchange chromatography. These 

separated fractions are then analyzed 

by reversed phase chromatography 

with MS/MS detection. 

A BioBasic SCX capillary column is 

used for the initial ion exchange 

separation. The columns have been 

designed to elute analytes with 

relatively low ionic strength buffer 

(salt) gradients, particularly for analysis 

of small organic molecules, nucleotides, 

peptides and small proteins. 

In addition, the columns are LC/MS 

compatible because volatile buffers 

figure 12. 2-Dimensional Protein Fractionation using BioBasic SCX and BioBasic18 Capillary Columns 

500mM 

NH 4 CO 2 H 

20mM NH 4 CO 2 H 

0mM NH 4 CO 2 H 

Full Sample 

can be employed at low ionic 

strength, often with volatile organic 

modifiers present. A BioBasic C18 

capillary column is used for the 

reversed phase separation, based on 

a 300Å silica to ensure accuracy in 

quantification. 

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The sample of protein digest is 

loaded onto the 100x0.32mm ID 

BioBasic SCX cation exchange 

column using a mobile phase of 

0.01% formic acid (0.1% for later 

fractions) at a flow rate of 2 µL/min 

(100:1 split flow) for a period of 20 

minutes. To elute the fractions onto 

the 100x0.18mm ID BioBasic 18 

reversed phase column, sequential 

20 µL injections of increasing 

concentrations of NH 4 CO 2 H (0 mM, 

20mM, 50mM, 100mM, 200mM, in 

0.1% formic acid) were used. Three 

of these fractions are shown in 

Figure 12. 

Sample: BSA Digest 

Fractionation from BioBasic SCX 

Column Dimensions: 100x0.32mm ID 

Inj. volume: 3µL (1500 fmol total digest on column) 

Fractions eluted with 20µL Ammonium Formate 

(concentrations listed, in 0.1% Formic Acid) onto 

100x0.180mm ID BioBasic 18 capillary 

Reversed Phase Separation of BSA Digest 

Mobile Phase: A: 0.01% Formic Acid 

B: ACN + 0.1% Formic Acid 

Flow: 2 µL/min 

Gradient: Time %B 

0 0 

3 0 

65 60 

Ionization Method: ESI+ 

Sheath Gas: 10 arb units 

Capillary Temp.: 130°C 

Spray Voltage: 2.5 kV 

Capillary Voltage: 12.00 kV 

Scan Range: 300-2000u 

11

Innovators in life and laboratory sciences, Thermo Electron Corporation provides 

advanced analytical technologies, scientific instrumentation, laboratory informatics 

solutions, and laboratory consumables to help scientists and clinicians to discover 

new drugs, improve manufacturing processes, and diagnose illness and disease. 

Unparalleled in our capabilities, we can help you every step of the way – from sample 

preparation and sample analysis through interpretation of results. 

For more information on our products and services, 

please visit our website at: www.thermo.com/hypersil-keystone 

Technical information contained in this publication is for 

reference purposes only and is subject to change without 

notice. Every effort has been made to supply complete and 

accurate information; however, Thermo Electron assumes no 

responsibility and will not be liable for any errors, omissions, 

damage, or loss that might result from any use of the 

information contained therein (even if this information is 

properly followed and problems still arise). 

Reference to specifications supersede all previous information 

and are subject to change without notice. 

www.thermo.com/hypersil-keystone 

©2003 Thermo Electron, Printed in USA 05/03. 

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