Vapor Phase Hydrolysis Procedu res

4. Vapor Phase Hydrolysis Procedu res

The vapor phase hydrolysis of protei n samples involves the placement of a small volume of acid i n the bottom of the Pierce vial reaction chamber and th ree cycles of evacuating and fl ushing with n itrogen to remove resid ual oxyge n . (Note: Acid is not added to the sample vials.) The evacuated reaction chamber is

then heated in the oven for the n u m ber of hours appropriate for the samples

( i . e . , 24 hours for proteins, Protocol A).

1 ) Place acid i n the bottom of the Pierce vial

Place 500 IJ I of 6N HCI i n the bottom of the Pierce vial . This solution will provide a saturated acid vapor environment d u ring hydrolysis, and m i n i m ize the evaporation of acid from the sample vials.

2 ) Place samples i n the Pi erce vial

U ncap the preweighed sample vials and insert them carefully i nto the Pierce vial reaction chamber with clean forceps. Cap the Pierce vial with the M i n ie rt valve l i d , and tighten it firmly. (The needle should sti ll be i n serted into the center bore of the valve, from step 2 of the Preparation Proced u res above.) For increased stability of the samples when the Pierce vial is hand led or placed i n the oven for h yd rolysis, it is a good precaution to place the reaction chamber i nto a small beaker.

3) Remove residual oxygen from the P ierce vial Slide the vacu u m tubing onto the 2 2 -gauge needle while the line is being flushed with n itrogen gas . Evacuate the Pierce vial until it reaches the target vacu u m noted d u ring the preparatory steps, and then flush the vial with n itrogen gas .

4) Repeat evacuation and nitrogen flushing cycle To rem ove residual oxyge n , repeat the evacuation and nitrogen flushing cycle

a total of three times. After the final evacuation, close off the reaction chamber while the vacu u m pump is sti l l p u m ping on the system . Q u ickly and carefully withdraw the need le from the bore of the valve, and i m med iately close the M i n iert valve. Turn off the pump and d i sconnect the vacuum tubing

from the reaction chamber.

5) Hydrolysis The evacuated Pierce vial reaction chamber can now be placed i n the p reheated oven for the d u ration of the hydrolysis (i . e . , 24 hours for the acid hydrolysis of proteins, Protocol A).

Vapor Phase Acid Hydrolysis in a Pierce Vial Reaction Chamber

1 63

6) Sample retrieval

When the evacuated Pierce vial has been in the oven for the prescri bed length of time, it can be removed with oven mitts and set aside a few

min utes to cool. After the M i n iert valve is carefully u n screwed and rem oved from the Pierce

vial, add 400 �I of 0 . 1 N Hel to each hydrolyzed sample. The samples should

be capped as soon as they are cool enough to handle. Store the samples i n t h e refrigerator until they can b e analyzed .

7) C lean u p

D ispose o f t h e acid i n t h e bottom of t h e Pierce vial b y pi petti ng it i nto a large volume of water (� 1 00 m l ) . Dispose of the d i l ute acid by pouring it down the sink u nder running water. Remove the silicone ru bber septum from the M i n iert valve and rinse the valve and the vial in deionized water to

remove traces of acid .

Protocol L