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APPENDIX
APPENDIX
Beware of extra-column band broadening
In recent years Tosoh has introduced several high efficiency column
types with small internal diameters. Examples are:
- 1 mm ID, 2 mm ID and 4.6 mm ID x 30 cm L TSKgel SuperSW3000,
- 4.6 mm ID and 6 mm ID x 15 cm L TSKgel SuperAW columns.
It is well known that when the column diameter decreases, peak
volumes decrease by the square of the ratio of column diameter.
In contrast, a decrease in column length results in a proportional
decrease in peak volume. Thus, when changing column dimensions
from 7.8 mm ID x 30 cm L to 6 mm ID x 15 cm L results in a reduction of
peak volume by a factor of (7.8/6)
2
*(30/15) = 3.4. Similarly, the reductions
in peak volume are 5.8 when going from 7.8 mm ID x 30 cm L to 4.6 mm ID
x 15 cm L, and 21.1 when replacing a 4.6 mm ID x 30 cm L column by one
that is 1 mm ID x 30 cm L. Such large reductions in peak volume require
that the HPLC system is optimized with respect to external factors
that contribute to the sample band broadening that takes place inside
the column. Neglecting to optimize the HPLC system can seriously
detract from the true column efficiency, which ultimately can result in
unacceptable analysis results.
Main contributors to extra-column band broadening are capillary tubing
that connect the column to the injector and the detector, injection
volume, detector cell volume, detector time constant, and others.
Separation Report 95 discusses some of the variables to check
when working with a smaller ID column, in this case the use of a 4.6
mm ID x 30 cm L TSKgel SuperSW3000 column (4 micron) compared
to a 7.8 mm ID x 30 cm L TSKgel G3000SW
XL
column (5 micron). You
can download this and other separation reports from our website:
APPENDIX B
Recommended standards for quality control of TSKgel
Columns
Approximate*
Standard
molecular weight (Da)
Adenylate Kinase
6,000
Alcohol Dehydrogenase
150,000
Aldolase
158,000
β
-Amylase
200,000
Blue Dextran
2,000,000
Bombesin
1,620
Bovine Serum Albumin (BSA)
67,000
Carbonic Anhydrase
29,000
α
-Chymotrypsin
25,200
α
-Chymotrypsinogen
25,700
Conalbumin
70,000
Cytidine
243
Cytidine-5’-monophosphate
323
Cytochrome C
12,400
D-Mannitol
182
Dopamine HCI
190
Enolase
67,000
Ethylene glycol
62
Ferretin
460,000
γ
-Globulin
150,000
Glutamate Dehydrogenase
55,000
Glycine Monomer
246
IgG
160,000
IgM
900,000
Insulin
6,000
Lactate Dehydrogenase
36,500
Lysozyme
14,500
Myoglobin
16,900
Ovalbumin
43,000
p-Aminobenzoic Acid
137
Peroxidase
40,200
Phosphorylase B
94,000
Polyethylene Glycol Kit
1.1K, 1.5K, 3.7K, 10.9K, 19.7K
Polyethylene Oxide Kit
18K, 39K, 86K, 145, 252K, 594K, 996K
Polystyrene Kit
530, 950, 2.8K, 6.2K, 10.3K, 15.7K, 43.9K,
102K, 186K, 422K, 775K, 1260K
Pyruvate kinase
58,000
Ribonuclease A
12,600
Thyroglobulin
660,000
Transferrin
80,000
Trypsin
23,300
Trypsin Inhibitor
20,000
Trypsinogen
24,000
Tryptamine•HCI
24,000
Uric Acid
168
* exact molecular weight will depend on the species