Tailor-made, customized columns for particular applications

 
In order to ensure the validity of HPLC results over an extended period of time,not only is the reproducibility of filling the column important but also to a high degree the batch-to-batch reproducibility of the packing material itself,whereby excellent resolution,respectively high efficiency and long stand times must be taken for granted. This is particularly true for the analysis of complex mixtures such as pesticides,polyaromatic hydrocarbons, amino acids,etc.For a range of selected applications,the time and effort of optimisation of the separation (selection of stationary phase,eluent composition,flow rate,temperature,etc.)may be eliminated simply by choosing the appropriate GROM special column. These columns are packed with stationary phases especially developed in our R&D laboratories for a particular application.The stationary phases are subjected to stringent quality control in order to guarantee the absolute reproducibility of their selectivity for the separation in question.Each special column is accompanied by a detailed protocol of the chromatographic conditions and,where applicable,the conditions of derivatization. GROM special columns are available in both the standard NovoGROM hardware or as NovoGROM cartridges for use with quick connectors in the sizes 2,3 and 4 mm internal diameter.(Please enquire for other dimensions,e.g. NovoGROM Microbore or NovoGROM capillary columns.)
I. Biochemical applications
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Button_mini_pink.gif (1036 Byte)High performance prepacked size exclusion chromatography column
Proteins and peptides
 
10 129 Peptide Separation
 
10129 Seite.gif (7226 Byte) 1) Oxytocin
2) Bradykinin
3) Angiotensin
4) Eledoisin
5) Neurotensin
6) Angiotensin I
Column phase:
Column size:
Eluent A:
B:
Gradient:
Flow rate:
Pressure:
Temperature:
Detection (UV):
Injection:		 GROM-Bio RP-1 (C18),5 µm
250 x 2 mm
0.1%TFA in H2O
0.1%TFA in H2O /ACN =30 /70
5 -100%B (0-30 min)
0.3 ml/min
16 MPa
RT
210 nm
10 µl (=10 pMol)
 
Stationary phase Field of application Order number
GROM-Bio Reversed Phase I
widepore C18		 small polypeptides,
nucleotides		 GS BR 1 0530 K 2502
250 x 2 mm cartridge
GROM-Bio Reversed Phase II
widepore C8		 peptides,medium
sized proteins		 GS BR 2 0530 K 2502
250 x 2 mm cartridge
GROM Bio Reversed Phase III
widepore C4		 proteins (MW 3 000 Dalton) GS BR 3 0530 K 2502
250 x 2 mm cartridge

 

High performance prepacked size exclusion chromatography column
Novarose™ SE - 100/17 for Size Exclusion Chromatography

Superior performance in purification, molecular weight determination and fast desalting of peptides and proteins

 
A new emulsification and crosslinking method has made it possible to modify highly purified agarose and to produce small,spherical beads, thereby decreasing pore size and particle size while keeping the matrix volume low.Thus,outstanding results are obtained employing Novarose SE in high performance size exclusion chromatography.Novarose ™ SE -100 / 17 columns are designed for analytical and semi-preparative separations of biopolymers such as proteins in the 10,000 to 100,000 Daltons molecular weight range.Due to the low diffusion coefficients involved,protein separation requirescomparatively low flow rates to give high resolution in size exclusion chromatography.This
is especially true when highly heterogeneous samples are applied. Nevertheless,impurities,which are always of interest but hidden at higher flow rates,can easily be detected at relative low flow rates.

Bild 21.JPG (6649 Byte)
 
10 184 Analytical separation of
protein mixture

10184 Seite.gif (8341 Byte)

 

Experimentals conditions:
column: Novarose TM SE --100 /17 - 300 x 8 mm; eluent: 0.05 M
Na-phosphate pH 6.7,0.15 M NaCl; flow rate: 0.25 ml /min; temperature: ambient; detection (UV): 214 nm; sample injected: mixture of 1) 24.6 µg thyroglobulin, 2) 24.6 µg aldolase, 3) 12.3 µg ovalbumin, 4) 12.4 µg carbonic anhydrase, 5) 24.0 µg ribonuclease and 25.0 µg insulin dissolved in 80 µl eluent buffer.
The following table summarizes the separation efficiency for carbonic anhydrase (peak 4)at different flow rates.Decreasing the flow rate clearly demonstrates the slower diffusion rate of larger molecules.The efficiency nearly doubles when reducing the flow rate by half.
10 186 Desalting of alcohol dehydrogenase
Data of resolution of proteins

 t1.gif (10904 Byte)*calculated on carbonic anhydrase,peak 4 (same experimental conditions as in Applic.10 184)

10185 Seite.gif (6967 Byte)
This figure represents an easy,quick method for the determination of molecular weights of globular proteins.The Kav values for the different proteins in the mixture are calculated and plotted against the logarithm of their molecular weight.Good linearity is shown in the recommended separation range.

Experimental conditions:
s.above, except 0.5 ml/min flow rate
10 185 Protein molecular weight determination
 
10186 Seite.gif (9476 Byte)
However,often speed is also considered as an important parameter in chromatographic procedures especially for less complex separations, such as the removal of salt from a biopolymer preparation or buffer exchange.A quick and simple desalting step is often required prior to enzymatic digestion.The rigidity and chemical stability of the Novarose SE also make it ideal for high flow rates or use with extreme pH values.Thus,the figure „desalting of alcoholdehydrgenase " shows the facile removal of 6 M guanidinum hydrochloride and dithiotreitol from alcohol dehydrogenase within a few minutes (flow rate 4,0 ml/min).