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Detection of Amino Acids Using HPLC with CD Detection
Amino acids are the "building blocks" of the body. Besides building cells and repairing tissue, they form antibodies to combat bacteria and viruses, are part of the enzyme and hormonal system, build nucleoproteins (RNA and DNA), carry oxygen throughout the body, and participate in muscle activity. The naturally occurring amino acids that comprise proteins are almost all of the levorotatory (L)- form. The L-form is the stereoisomer that rotates plane-polarized light to the left. One method to detect amino acids and determine their stereoisomers is with HPLC.
Introduction
Chirality describes the ability of a molecule to rotate the plane of polarized light either to the right or to the left. All of the amino acids in proteins exhibit the same absolute steric configuration in that they are all L-α-amino acids. D-amino acids are not found in proteins, although they exist in nature.
click the image to enlarge
Figure 1. HPLC separation of Pro, Ser, Thr, and Cys amino acids (top to bottom).
| The role that amino acids play is crucial to the activity of many reactions. In solution, the amino acid R-groups dictate the structure-function relationships of peptides and proteins. It is the amino acid R-groups that allow enzyme reactions to occur. Equally important is the ability of histidines in hemoglobin to buffer the H+ ions in red blood cells. It is this property that allows hemoglobin to exchange O2 and CO2 at the tissues or lungs, respectively. The aromatic R-groups in amino acids absorb ultraviolet light with an absorbance maximum in the range of 280 nm. The ability of proteins to absorb ultraviolet light is due to the tryptophan that strongly absorbs UV. This note describes chiral detection of amino acids with HPLC.
Experimental
Amino acids were examined using a Jasco HPLC with a CD-2095 detector. The separation was accomplished with a CROWNPAK CR(+) (4.0 mm I.D. × 150 mm/L) column. The eluent was HclO4 with flow rates of 0.4 to 0.8 mL/min. The column temperature varied from 0 to 25°. Injection volume was 10 μL (2 mg/mL, 20 μg). Amino acids were also separated following precolumn derivatization with DABS-Cl. For these separations, a CrestPak C18S column was used with a mixture of: A) 8mM Sodium Dihydrogenphosphate Dihydrate in H2O with 4% Dimethyl formamide (DMF), and B): Acetonitrile (ACN). Temperature was 40 C, injection volume 10 μL, and flow rate 1 mL/min. The gradient is shown below.
| Time (min) |
A (%) |
B (%) |
Time |
A (%) |
B (%) |
| 0 |
85 |
15 |
16 |
49 |
51 |
| 9 |
70 |
30 |
18 |
46 |
54 |
| 14 |
59 |
41 |
23 |
10 |
90 |
Results and Discussion
Figures 1 shows the HPLC separation of proline, serine, threonine, cysteine, lysine, and arginine without precolumn derivatization. Each compound exhibited both a positive and negative peak in the CD verifying the presence of both enantiomers. In the case of molecules like Isoleucine (not shown) that have two chiral centers, two separate sets of peaks are found, one set for each chiral center.
click the image to enlarge
Figure 2. Separation of Cysteine with DABS-CL.
| The separation of cysteine (Figure 2) was also accomplished following precolumn derivatization with DABS-Cl. In this case, pre-derivitization was required for detection because a chiral column was not used. Derivatization sharpens the peaks and allows a more complete separation. Regardless of whether the separation is performed with a chiral column or with precolumn derivatization, Jasco's CD detector can offer sensitive and selective detection of chiral compounds such as amino acids.
Jasco Inc.
8649 Commerce Drive
Easton, MD 21601
ow
JASCO Incorporated
8649 Commerce Drive
Easton, MD, 21601
|
