Creatine Case Study

Case Study

History

Learning Goals /
Concept Map


Creatine and Related Compounds

Structure

Amino Acids

Creatine in the Body

Equilibrium

Creatine-Creatinine Equilibrium

Creatinine Test for Kidney Function

Detection

Regulation and Ethics

Amine & Nitrile Chemistry

Laboratory Synthesis

Chemical Analysis

Creatine-Phosphocreatine Equilibrium

Uses & Side Effects


Chemical Analysis

After synthesizing creatine hydrate in the laboratory, we can use various analytical techniques to ascertain that we have our desired product.  Here we will consider three types of analysis:

  • melting point (MP)
  • thin layer chromatography (TLC)
  • nuclear magnetic resonance (NMR)

Melting Point (MP)
Melting point provides information about the purity of a solid sample.  The term “melting point” is a bit of a misnomer because the temperature at which a substance melts is often reported as a range.  A small temperature range is desirable; this may be obtained by slow, even heating of the sample.  If the sample is heated too rapidly during the course of melting point determination, the range may be large.  A large melting range can also indicate the presence of impurity in the sample.  Impurities may also result in a melting point that is lower than the expected value (when determining the melting point of a known compound).

As we consider the types of information we can gain from thin layer chromatography and nuclear magnetic resonance, it will be useful to remember the reaction we use to synthesize creatine in the lab and to look at the structures of cyanamide, sarcosine, and creatine monohydrate:

Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) is a simple analytical technique that allows us to differentiate compounds on the basis of polarity.  It is often used by organic chemists to determine whether a synthetic reaction has gone to completion and whether the product is pure.

Nuclear Magnetic Resonance (NMR)
Proton nuclear magnetic resonance (1H NMR) spectroscopy is an analytical technique that allows us to create a “map” of the carbon-hydrogen framework of a molecule—that is, the connectivity of the atoms and functional groups in the molecule.  An 1H NMR spectrum provides a wealth of information that allows us to elucidate molecular structure:

  • The number of peaks tells us how many different types of equivalent protons exist in a molecule.
  • The position of each peak (also called the “chemical shift”) gives us information about the electronic environment of each proton in the molecule—in other words, the types of atoms and functional groups (electron-donating or electron-withdrawing) that surround each proton in the molecule.
  • The integration of each peak tells us the number of equivalent protons that are present in the same molecule.
  • The number of lines into which each peak is split (also called the “spin-spin splitting”) tells us how many neighboring protons each proton in the molecule has.

How can we use 1H NMR to determine whether we have made creatine from cyanamide and sarcosine?  Let’s again consider the reaction between cyanamide and sarcosine to produce creatine:

Proton Equivalence
Let’s use the number of different types of protons in cyanamide, sarcosine, and creatine to predict the maximum number of peaks present in each 1H NMR spectrum.

Chemical Shift
The chemical shift of each peak is determined by the nature of the atoms and functional groups surrounding each proton in the molecule.  The presence of electron-withdrawing groups causes a downfield shift (to the left of the NMR spectrum), whereas the presence of electron-donating groups causes an upfield shift (to the right of the NMR spectrum).  These effects are additive, so two electron-withdrawing groups cause a greater downfield shift than just one electron-withdrawing group.  A summary of these effects is shown below. 


(from Bruice, Organic Chemistry, 3rd ed., Prentice Hall: Upper Saddle River, NJ, 2001, p 543)

Integration
The integration tells us the relative number of protons represented by each peak in the 1H NMR spectrum.  We often use the integration to determine the ratio of protons in different peaks of the spectrum.

Spin-Spin Splitting
We use the splitting patterns of each peak to tell how many “neighboring” protons each proton has.  In molecules with single bonds, neighboring protons are separated by three single bonds.

Splitting patterns follow the “n+1 rule”, which states that a peak with n neighbors should be split into n+1 lines in the 1H NMR spectrum.

Summary
We have seen that melting point and thin layer chromatography both provide information about the purity of the product of an organic synthesis and that proton nuclear magnetic resonance gives insight into chemical structure.  By using a combination of analytical techniques, we are able to determine the outcome of an organic synthesis.