Detergent Removal Using Amberchrom CG161 - - Chromatography Online
FindAnalytichem Custom Search
About Search
Detergent Removal Using Amberchrom CG161

Introduction


Figure 1: Structure of Triton X-100.
Detergents are commonly used in hydrophobic cell membrane protein purification and viral removal/inactivation. These detergents must eventually be removed during the purification process. If a detergent has low critical micelle concentration (CMC), it can be difficult to remove from solution using techniques such as dialysis or gel filtration. Therefore, a detergent with a low CMC like Triton X-100 requires different removal techniques. Triton X-100 is a non-ionic detergent with an approximate molecular weight 625 and CMC value of 0.24 mM. The structure of Triton X-100 is shown in Figure 1.


Figure 2: Dynamic binding capacity of Amberchrom CG161 for cephalosporin C and BSA.
Amberchrom CG161 was chosen as a chromatographic solution for the removal of Triton X-100. It is a polystyrene divinylbenzene polymer used for the purification of peptides and antibiotics. This resin has been used in commercial purification processes for several decades and is an excellent choice for detergent removal because of its unique pore structure and hydrophobic nature. As shown in Figure 2, Amberchrom CG161 has a high capacity for small molecules such as cephalosporin C (479 MW), and virtually no capacity for larger proteins such as BSA (67000 Da). Therefore, detergents should effectively be removed from protein feedstocks using Amberchrom CG161.

Results and Conclusions


Table 1: Capacity comparison for Triton X-100.
For detergent capacity experiments, Amberchrom CG161 was compared with another commercially available resin which is sold specifically for detergent removal, SDR HyperD (Pall Life Sciences, East Hills, New York, USA). Amberchrom CG161M and SDR HyperD were packed into separate glass 1.0 cm ID × 7.0 cm L columns and the columns were used to determine the dynamic binding capacities for Triton X-100. A phosphate buffered solution containing 5 mg/mL of Triton X-100 was passed through the column at a flow-rate of 2 mL/min (152 cm/hr) and the effluent was collected. After complete saturation of the column (200 mL of effluent collected), the experiment was terminated. The 1% breakthrough capacity and total capacity were then calculated.

Conditions:
Column: 1cm ID × 7cm L
Flow-rate: 2 mL/minute
Sample: 5 mg/mL Triton X-100
Mobile phase: Sodium phosphate buffer, pH 7.4
Detection: UV @ 280nm

As can be seen in Table 1, Amberchrom CG161M demonstrated higher 1% and total capacity values for Triton X-100 as compared to SDR HyperD. Amberchrom CG161 chromatographic resin is an effective tool for the removal of detergents from biological samples. It provides high capacity for non-ionic detergents such as Triton X-100.

ADVERTISEMENT

Rate this page
Your original vote has been tallied and is included in the ratings results.
View our top pages
 Average rating for this page is: 2.79
post a comment
Please complete the form below to post your comment.
Bold = Required
Email Address Your email address will NOT be published.
Display Name appears with your comment
City
State
Country
Remember me: YesNo
Comment read our privacy policy
Note: does not support HTML
All Comments submitted are subject to review, and may be delayed before posting. Comments will be posted at the discretion of the editor.
LCGC E-mail Newsletters
Select each newsletter you wish to subscribe for:
| Monthly
| Weekly
| Monthly
 Name:
 E-mail:
more info
Survey
What was your primary reason for attending HPLC 2009?
Education — furthering knowledge by attending lectures
Attending exhibition
Exhibiting at the show
A combination of all three
Other
Education — furthering knowledge by attending lectures
0%
Attending exhibition
0%
Exhibiting at the show
67%
A combination of all three
0%
Other
33%
View Results
Tell us more about what you thought of HPLC in our LinkedIn group
Source: LCGC Europe, 12/2/2005
Click here