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Fig. 2. Relative elution positions of hydroxylysyl-pyridinoline, (Hyl-Pyr); lysyl-
pyridinoline (Lys-Pyr); isodesmosine (I-DES); desmosine (DES); histidino-
hydroxylysinonorleucine (HHL); dihydroxylysinonorleucine (DHLNL); and
hydroxylysinonorleucine (HLNL) on an ion-exchange amino acid analyzer using
modified buffers. (DHLNL and HLNL are the sodium borohydride reduction products
of hydroxylysino-ketonorleucine, HLKNL and dehydro-hydroxylysinonorleucine,
∆
-HLNL, respectively).
3.7. HPLC Techniques
1.
After hydrolysis and lyophilization each sample is rehydrated in an acidic, aque-
ous solution at a concentration of approximately 1
µ
g of collagen per
µ
L. The
HPLC system used for analysis dictates the solvent used for rehydration.
2.
This laboratory originally used a 250
m, octadecyl silane (ODS)
column eluted with a 5-35% acetonitrile (MeCN) gradient in water at 1 mL/min
over 70 min (0.5%/mL/min) (a modification of
(4
). Both aqueous and organic
solvents contained 0.05
M
heptafluorobutyric acid (HFBA), ion pairing agent.
Samples destined for this system were hydrated in 5% or 10% HFBA (
see
Note 21
).
×
4.6 mm, 5
µ
3.
Currently, we use a Shandon (Runcorn, UK) Hypercarb S, 100
4.6 mm, gra-
phitic carbon column eluted with a 0 - 12% tetrahydrofuran (THF) gradient in
water at 1 mL/minute. Both aqueous and organic solvents contain 0.5%
trifluoroacetic acid (TFA). Samples destined for analysis by this system are
hydrated in 1% TFA (
see
Note 22
).
×
4.
Following rehydration the samples are 0.2
m filtered into tapered glass sample
vials (Chromacol, Welwyn Garden City, UK), sealed and stored at 4
µ
°
C. An
aliquot of up to 90
L is loaded onto the analytical column via an autosampler or
a larger volume can be manually loaded via a Rheodyne valve with a 500
µ
µ
L
sample loop (Anachem, Luton, UK).
5.
Prior to use the HPLC buffers are degassed either under vacuum (for 10 min) or
by helium sparging (1-2 min).
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