How big is big?

[sheanhung]

I have bought some pure gelatin solid, for information, gelatin is protein.

 

My question is how big is the protein polymer? I want to know it because i plan to dissolve it in water and introduce it into HPLC ( high performance liquid chromatography).

 

Basically, the analyte molecules introduced into the HPLC column shouldn't be too big, otherwise the molecules will stuck inside the column. But the references didn't mention how big is the analyte is consider "big" to column? The term 'big' is so abstract.

[RyanJ]

Thats a big question.

 

As with any protein the composition can vary from one too another, this makes it very hard to measure its composition with any degree of accuracy.

 

Research shows that its approximate composition is

 

The approximate amino acid composition of gelatin is: glycine 21 %' date=' proline 12 %, hydroxyproline 12 %, glutamic acid 10 %, alanine 9 %, arginine 8%, aspartic acid 6 %, lysine 4 %, serine 4 %, leucine 3 %, valine 2 %, phenylalanine 2 %, threonine 2 %, isoleucine 1 %,hydroxylysine 1 %, methionine and histidine <1% and tyrosine < 0.5 %. These values vary, especially the minor constituents, depending on the source of the raw material and processing technique.

[/quote']

 

From this you could probably work out the M_r and such but its not a very accurate measurement in this instance.

 

-- Ryan Jones

[John Cuthber]

Why do you want to put gelatine down an HPLC?

[sheanhung]

Why do you want to put gelatine down an HPLC?

 

I plan to analyse the fingerprint of different gelatins in HPLC.

 

My supervisor suggested me that i should hydrolyse the gelatins into amino acids before they can be introduced into HPLC or HPLC-MS. I guess it is not an easy experiment for a degree student like me.

[RyanJ]

My supervisor suggested me that i should hydrolyse the gelatins into amino acids before they can be introduced into HPLC or HPLC-MS. I guess it is not an easy experiment for a degree student like me.

 

Well that would be a much simpler method. if you can hydrolyse the polypeptide, in conc. sulphuric acid for about a day and identify the constituents you should be able to work out the Mr of the original polypeptide quite easily

[sheanhung]

Well that would be a much simpler method. if you can hydrolyse the polypeptide, in conc. sulphuric acid for about a day and identify the constituents you should be able to work out the Mr of the original polypeptide quite easily

 

I used 6M HCl to hydrolyse gelatin at 150 degree celcius. I tried to introduce the hydrolysed amino acids into LC-MS system using C18 column, and mobile phase (A & B) A= 0.2% formic acid in water, B= 0.2% formic acid in acetonitrile. I used gradient method, i.e. 2-60% B in 60 minutes.

 

However, most of the amino acids eluted out from the column within 5 minutes. I guess the amino acids are very polar and they are not well retained by the column.

 

If I'm still using C18 column, what mobile phases are more suitable? or what else should i reconsider?

 

[John Cuthber]

There are about a trillion hits on Google for "amino acid analysis" HPLC. Most of them involve reacting the amino acids with something to make them easier to detect and less polar.

This one seems pretty typical.

http://www.canr.msu.edu/comparativenutrition/Amino%20Acid%20analysis%20of%20Hydrolysates.doc

I can't say I've tried it but it looks like a good place to start.

Of course, that's OK for a UV or fluorescence detector. LC/MS can be a bit fussy. You will need a volatile buffer rather than sodium acetate and you might need some clean-up stage.

[sheanhung]

cool.... thanks!