<p class="jove_content">חבר מומן על ידי בריטניה מחקר לאגודה פרקינסון (גרנט F1002). עבודה זו נתמכה על ידי MRC החדש החוקר במענק מחקר (מר/L010933/1), על-ידי תוכנית MRC להעניק מר/N026004/1 ל- PAL, ועל ידי BBSRC studentship BB/M017222/1 למטוס. אנו מודים על עוקבה 2014-15 של MPharm חלק 1 סטודנטים מאוניברסיטת רדינג עבור השתתפות איטרציה הראשון של גדודים הזה מעשי, ובעקבות משוב, קלט.</p>

<p class="jove_content">הערה: להלן הפרוטוקול פותחה שיתקיים במשך שעתיים (כולל השלמת גליונות עבודה ב- class) במעבדה הוראה בנוי למטרה. המדרגות ניסיוני היו בכוונה לשים יחד כדי לא לכלול ניתוח מפורט יותר של קינטיקה אנזימטית (המבוצע במסגרת קורס זה באמצעות מודל נתונים); עם זאת – כאמור בדיון – הפרוטוקול מספק תבנית עבור ניתוחים מתקדמים יותר בהתאם רמת הפרי זמן ומתקנים, סטודנט.</p>
<p class="jove_content">בטיחות: מעשית זו צריכה להתבצע על פי הכללים של טוב כימי מעבדת האימון (GCLP) — ציוד מגן אישי, כולל מעבדה מהודק מעיל, כפפות חד פעמיות, בטיחות משקפיים צריכים ללבוש כל הזמן במעבדה .</p>
<p class="jove_title">1. חילוץ האנזים</p>
<ol><li>למחוץ לקטאז טבליה אחת, המכילה 200 מ ג של אנזים, אבקת אפילו באמצעות ומכתש.</li>
	<li>מקם את האבקה המתקבלת לתוך צינור 15 מ"ל מתויג"ההשעיה", resuspend ב- 10 מ ל תמיסת פוספט buffered (PBS) של 100 מ מ.</li>
	<li>מערבולת עבור 1 דקות למקסם את שאיבת אנזימים.</li>
	<li>להעביר 1 מ"ל מ ההשעיה לתוך צינור צנטריפוגה 1.5 mL צנטריפוגה עבור 1 דקות ב x 10,000 <em>ג'י </em>לחלקיקים מוצקים משקעים.</li>
	<li>העברת µL 500 של תגובת שיקוע צינור נקי mL 1.5 מתויג "לקטאז להוצאת".</li>
</ol><p class="jove_title">2. התבוננות התגובה ערכי צבע מוחלטים</p>
<ol><li>µL במקום 390 מ"מ PBS לתוך צינור 1.5 mL מתויג"תגובה A".</li>
	<li>להוסיף 100 µL של 5 מ מ ONPG פתרון, לערבב היטב על ידי vortexing.<br>התראה: מעשית זו משתמשת אורתופדיה-Nitrophenol -<em>beta</em>-D-galactopyranoside (ONPG) כתחליף לקטוז. מאחר ONPG הוא מתחם בעל תאים פנוליים, זה צריך להיות מטופל בזהירות. במקרה של מגע עם העור עם ONPG העור חשוף צריך לרחוץ באופן מיידי. נשפך כל שצריך למחות מיד עם מגבות נייר כדי להיות מסולק לתוך זרם פסולת המתאים.</li>
	<li>להוסיף 10 µL של תמצית לתוך צינור התגובה A ו לערבב היטב על ידי vortexing.</li>
	<li>לצפות את תערובת התגובה למשך 5 דקות, הערה כל שינוי ערכי צבע מוחלטים לפתרון.</li>
</ol><p class="jove_title">3. מדידת פעילות אנזים</p>
<ol><li>להגדיר את שני צינורות 1.5 mL: אחד מתויג "התגובה B", אחד שכותרתה "שליטה".</li>
	<li>להוסיף µL 390 מ"מ PBS התגובה B שפופרת, µL 400 מ"מ PBS אל הצינור שליטה ולאחר מכן להוסיף 100 µL של 5 מ מ ONPG כל שפופרת.</li>
	<li>מערבבים את התוכן על-ידי vortexing.</li>
	<li>להוסיף 10 µL של תמצית לקטאז הצינור התגובה B, לערבב על ידי vortexing ולאפשר את התגובה להמשיך עבור 1 דקות בטמפרטורת החדר.</li>
	<li>ברגע 1 דקות חלף, להוסיף 500 µL של 1 מ' סודיום קרבונט שני צינורות כדי לעכב את האנזים לקטאז על ידי הגדלת ה-pH, ובכך מסיימת את התגובה.</li>
	<li>העבר 500 µL כל שפופרת לתוך וואקום ספקטרופוטומטרים נקי ולמדוד את ספיגת-420 nm באמצעות ספקטרופוטומטרים את.</li>
	<li>הערה ספיגת עבור התגובה B ודגימות הבקרה ולאחר מכן לחסר את ערך הפקד מהערך תגובת להפיק בשינוי ספיגת עקב הידרוליזה של ONPG בנוכחות האנזים פעיל.</li>
</ol><p class="jove_title">4. השפעת הטמפרטורה על פעילות האנזים</p>
<ol><li>להגדיר פקד שלושה צינורות וצינורות התגובה שלושה כמתואר בסעיף 3, תווית אלה<strong> </strong>"4 ° C", "חדר Temp" ו- "37 ° C".</li>
	<li>בעקבות תוספת של אנזים לחלץ, דגירה שפופרת אחת על קרח, אחד בטמפרטורת החדר, ואחד ב 37 מעלות צלזיוס (באמבט מים מחומם מראש).</li>
	<li>לאפשר את תגובות המשך עבור 1 דקות, ואז לסיים את התגובה על-ידי הוספת µL 500 של 1 מ' סודיום קרבונט כל שפופרת.</li>
	<li>למדוד את ספיגת-420 nm לכל שפופרת כמתואר בסעיף 3. להקליט את הערכים, לחסר את ערך הפקד מהערך התגובה בכל מקרה ומקרה.</li>
</ol>

<ol>
	<li>Berg, J. M., Tymoczko, J. L., Gatto, J. G., Stryer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Biochemistry</em>. , (2015).</li><li>Jittam, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+seaweed+enzyme-catalyzed+bromination+of+bromophenol+red:+An+inquiry-based+kinetics+laboratory+experiment+for+undergraduates.">Red seaweed enzyme-catalyzed bromination of bromophenol red: An inquiry-based kinetics laboratory experiment for undergraduates.</a> <em style='font-style: italic'>Biochemistry and Molecular Biology Education</em>. <b>37</b>, 99-105 (2009).</li><li>Troelsen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult-type+hypolactasia+and+regulation+of+lactase+expression.">Adult-type hypolactasia and regulation of lactase expression.</a> <em style='font-style: italic'>Biochimica et biophysica acta</em>. <b>1723</b>, 19-32 (2005).</li><li>Juers, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+view+of+the+action+of+Escherichia+coli+(lacZ)+beta-galactosidase.">A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.</a> <em style='font-style: italic'>Biochemistry</em>. <b>40</b>, 14781-14794 (2001).</li><li>Plimmer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+presence+of+lactase+in+the+intestines+of+animals+and+on+the+adaptation+of+the+intestine+to+lactose.">On the presence of lactase in the intestines of animals and on the adaptation of the intestine to lactose.</a> <em style='font-style: italic'>Journal of Physiology</em>. <b>35</b>, 20-31 (1906).</li><li>Krebs, H. A., Salvin, E., Johnson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+citric+and+alpha-ketoglutaric+acids+in+the+mammalian+body.">The formation of citric and alpha-ketoglutaric acids in the mammalian body.</a> <em style='font-style: italic'>The Biochemical journal</em>. <b>32</b>, 113-117 (1938).</li><li>Vesa, T. H., Marteau, P., Korpela, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactose+intolerance.">Lactose intolerance.</a> <em style='font-style: italic'>Journal of the American College of Nutrition</em>. <b>19</b>, 165S-175S (2000).</li><li>Robayo-Torres, C. C., Nichols, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+differentiation+of+congenital+lactase+deficiency+from+adult-type+hypolactasia.">Molecular differentiation of congenital lactase deficiency from adult-type hypolactasia.</a> <em style='font-style: italic'>Nutrition Reviews</em>. <b>65</b>, 95-98 (2007).</li><li>Swallow, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+lactase+persistence+and+lactose+intolerance.">Genetics of lactase persistence and lactose intolerance.</a> <em style='font-style: italic'>Annual Review of Genetics</em>. <b>37</b>, 197-219 (2003).</li><li>Enattah, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+introduction+of+two+lactase-persistence+alleles+into+human+populations+reflects+different+history+of+adaptation+to+milk+culture.">Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture.</a> <em style='font-style: italic'>American Journal of Human Genetics</em>. <b>82</b>, 57-72 (2008).</li><li>Lowe, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rapid+detection+of+lactose+fermentation+in+paracolon+organisms+by+the+demonstration+of+beta-D-galactosidase.">The rapid detection of lactose fermentation in paracolon organisms by the demonstration of beta-D-galactosidase.</a> <em style='font-style: italic'>Journal of Medical Laboratory Technology</em>. <b>19</b>, 21-25 (1962).</li><li>Russo, S. F., Moothart, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+study+of+the+Enzyme+Lactase.">Kinetic study of the Enzyme Lactase.</a> <em style='font-style: italic'>Journal of Chemical Education</em>. <b>63</b>, 242 (1986).</li></ol>

<p class="jove_content">המחברים יש שאין ניגודי אינטרסים לחשוף.</p>

<p class="jove_content">נתונים היסטוריים הידע וההבנה של אנזימים, תגובות אנזימטיות, קינטיקה אנזימטית נדרש עבור מגוון רחב של נושאים המתפרסות על-פני הביולוגיה, וההתערבות ו פרמקולוגיה. הפרוטוקול המתואר לעיל משתמש לקטאז כדוגמה של אנזים הרלוונטיים לבריאות האדם על הפגנת אנזימטיות, מתן מפתח מיומנויות שיכול לשמש בניסויים מבוסס מעבדה מתקדמים יותר.</p>
<p class="jove_content">פרוטוקול זה תוכנן בכוונה להיות הכי חזקים כמו אפשרי, דבר המאפשר לתלמידים עם מעט או ללא ניסיון מעבדה רטוב לתוצאות interpretable בזמן קצר של זמן. בשנת 2014/2015 הלימודים בביה ס מאוניברסיטת רדינג של בית מרקחת, סך של 144 סטודנטים, מאורגנת בקבוצות של 3, ביצעו את הניסוי "מדידת לקטאז", עם מסוגלים לזהות רמה מסוימת של פעילות אנזימטי של לקטאז tablet כל הקבוצות תמציות.</p>
<p class="jove_content">ישנם מספר צעדים קריטיים של פרוטוקול זה. קודם כל, זה חיוני כי החילוץ הראשוני הוא יעיל, ומאפשר solubilization של האנזים פעיל מספיק עבור ניתוחים שלאחר מכן. מניסיוננו, בשנה הראשונה סטודנטים ניתן להשיג זאת על-ידי ביצוע הפרוטוקול כמתואר; עם זאת הדרכה של מפגינים מעבדה נדרשה בשלב זה. למרות נראה נקודה מובנת מאליה, אחד מגורמי ההצלחה של פרוטוקול זה הוא היכולת למדוד באמצעי אחסון, כראוי בתווית צינורות ובזהירות ההוראות. בעוד רמה מסוימת של מיומנות ניתן להניח עבור סטודנטים רבים, פיקוח קפדני, בקשה ברורה לתלמידים לבקש סיוע בכל מקרה שבו הם בטוחים מה לעשות הבא מספק את הסבירות הגדולה ביותר של assay מוצלחת.</p>
<p class="jove_content">הערכת התלמיד יכול להתבצע על ידי ב- class בגליון העבודה (זמין כחומר משלים), מבקשים התלמידים לציין נתוני הניסויים, תגובה על התוצאות שלהם קישור לקריאת/רקע מידע מחומר ההרצאות, או יותר מפורט, מתוך הערכה בכיתה עם מודל נתונים שסופקו המאפשר ניתוח קינטי להיות ניסיון ולא מוערכים.</p>
<p class="jove_content">פרוטוקול שהוצגו במסמך זה הוא דוגמה פשוטה assay פעילות לקטאז תחת תנאי מעבדה מלמד, קואנקה כדי להגדיל את המורכבות של הניסוי כדי לאפשר ניתוח מתקדם יותר. בפרט, פרוטוקול שהוצגו מספקת מבוא לרעיון של קינטיקה אנזימטית אך אינו מאפשר ניתוח קינטי של נתוני הניסוי. ככזה, אנו ממליצים לצפייה הפרוטוקול המתואר כמו תבנית הראשוני עבור מחלקות, עם הפרטים המדויקים הותאם שיתאים את מטרות ספציפיות ואת כישוריו של קבוצה סטודנט התחייבות הניסויים. דוגמאות לניסויים המורחבת יכול לכלול: לחזור על המדידות בטמפרטורות שונות, באמצעות ריכוז סובסטרט שונים כדי לאפשר ניתוח סטטיסטי, קינטי ניתוח של נתונים (מתאים עבור סטודנטים ביוכימיה/מקצועות ראשיים), החל מספר לוחות שונים, המאפשר את התלמידים להעריך את פעילות שונים של האנזים בכל אחד (עם התוספת של שליטה טבליות, מתאים למדע פורנזי סטודנטים/מקצועות ראשיים), הערכת ההשפעה של חום דנטורציה או נושאת את המתחרים ניסויים על-ידי תוספת של לקטוז או מעכבים של לקטאז (<strong>טבלה 1</strong>). פרוטוקול מפורט לניתוח קינטי של לקטאז בעבר כבר פורסם<sup class="xref">12</sup>.</p>
<p class="jove_content">כוח חשוב של פרוטוקול זה הוא כי הוא משלב הקדמה בסיסי קינטיקה אנזימולוגיה, האנזים עם חקר מקרה אמיתי של אנזימולוגיה ברפואה. זה הופך את המעבדה. ניסוי הרלוונטיות לסטודנטים לרפואה, בית מרקחת, ביו ונושאים קשורים נוספים. חשוב מכך, העובדה כי יש בעיה רפואית רצינית וגם בעיית תזונה נפוצה יותר המשויכים לקטוז קטבוליזם מספק הזדמנות להעלות מגוון רחב של בעיות רפואיות ואתיות עם התלמידים. זה יכול לכלול את הנושאים בדיקה גנטית לטיפול בבעיות רפואיות, הקשורים דיונים הנוגעים ריפוי גנטי ייעוץ גנטי, כמו גם שימוש ואת מכירת צמחי מרפא תוספי מזון. מגבלה אחת גדולה היא כי הפרוטוקול אינו מאפשר הערכה מדויקת של ריכוז אנזים, עקב החומר ההתחלתי המשמש להפקת. שינוי להביא בחשבון זה, עם זאת, יהיה לשימוש ריאגנט כיתה אנזים וזמינותו. חשוב, זה יאפשר גם לחישוב מדויק יותר של קינטיקה של האנזים לקטאז.</p>
<p class="jove_content">לסיכום, assaying את הפעילות של לקטאז במעבדת ההוראה מספק סביבה חזקים, מרתק ומעניין הקדמה השדה של ביולוגיה אנזים בשלב מוקדם סטודנטים.</p>

<p class="jove_content">אנזימים הם סוג מיוחד של חלבון זה לשמש זרזים ביולוגיים עבור תגובות כימיות חי אורגניזמים<sup class="xref">1</sup>. הפעולה של אנזימים חיונית לחיים, לאספקת אנרגיה, סילוק פסולת, ומאפשר אורגניזמים לתפקד. ההבנה אנזימים, לפיכך, הוא קריטי עבור הבנה מלאה של החיים. ידע כזה חיוני עבור מגוון רחב של תוכניות ברמת תואר באוניברסיטה, החל מדעי הרפואה לביולוגיה. בעוד רקע מפורט ועד עקרונות זרז מודלים תיאורטיים של פעילות אנזים יכולה להינתן לתלמידים באמצעות הרצאות, חומר קריאה, המאפיינים של אנזים תגובות הכי מובנת על ידי ידיים על ניסיון מעשי של אנזימים בפעולה כאמור הפגינו<sup class="xref">2</sup>. פרוטוקול זה מספק פשוט לעקוב פרדיגמה נסיונית למדידת פעילות אנזים בתנאי מעבדה, לקטאז כדוגמא של אנזים עם פעילות הנוגעים לתזונה אנושית ובריאות.</p>
<p class="jove_content">ההידרולז glycosidic לקטאז (EC 3.2.1.23/26) הוא אנזים חשיבות התזונה מרכזית עבור יונקים<sup class="xref">3</sup>. הפעילות של לקטאז מאוד כולו דרך האבולוציה, ונגזרת ממשפחת בטא-galactosidase של אנזימים – משפחה מתנת <em>Escherichia coli</em> דרך<em> הומו ספיינס</em> (<strong>איור 1</strong>, PDB 1JZ8)<sup class="xref">4</sup>. חשיבות קריטית לקטאז באווירה התזונה האנושית נובעת תפקידה המאפשר פירוק של לקטוז למרכיביו בוחרים חד-סוכר, אשר לאחר מכן ניתן להשתמש ליצירת אנרגיה בגוף. לקטאז ה מזרז הידרוליזה של קשר גליקוזידי ב דו-סוכר חלב (לקטוז), שחרור גלקטוז וגלוקוז (<strong>איור 2</strong>)<sup class="xref">5</sup>. Monosaccharides אלה משמשים אז בעיקר לדור של אדנוזין טריפוספט (ATP) <em>דרך </em>מעגל קרבס, זרחון חמצוני<sup class="xref">6</sup>. במהלך neonate והתפתחות תינוקות, לקטאז מתבטאת במיוחד מערכת העיכול האנושית, פירוק לקטוז קיבל חלב של לקטוז אילו הוא הרכיב העיקרי פחמימות, ואחד מקורות מרכזיים של תזונה במהלך השנים הראשונות<sup class="xref"> 7</sup>. החשיבות הרפואית של לקטאז מודגשת על ידי מולדת לקטאז (המחלקה לחקירות), מצב נדיר הקרים autosomal נגרמת על ידי מוטציות בגן לקטאז (<em>LCT</em>) קידוד האנזים לקטאז<sup class="xref">8</sup>. תינוק בן יומו תינוקות עם המחלקה לחקירות להפגין מעט מאוד פעילות לקטאז, ובכך הם אינו ניתן להזנה על חלב אם, בכל סוג אחר של חלב, או נוסחה המכילה לקטוז.</p>
<p class="jove_content">במהלך הילדות, ביטוי לקטאז מוקטן בדרך כלל; עם זאת, הפחתה זו בעקבות הגמילה משתנה מבחינה גיאוגרפית, כ- 35% של מבוגרים ברחבי העולם ממשיכים להביע את האנזים<sup class="xref">9</sup>. ביטוי מתמשכת של לקטאז, המכונה התמדת לקטאז, מאפשר ליחידים להמשיך לעכל חלב ומוצרי חלב ממגוון מקורות. לעומת זאת, האובדן של לקטאז הביטוי יכול להוביל אי סבילות ללקטוז, המכונה גם למבוגרים hypolactasia (ATH), הנובע חוסר היכולת לפרק לקטוז בבטן. ATH מאופיין על ידי תהליך בנייה למעלה של לקטוז במעי הגס מבליעת לקטוז המכילה מוצרי מזון. במעי הגס, מותסס של לקטוז שנצבר על ידי החי והצומח מיקרוביאלית מעיים, שחרור גזים כולל מימן, מתאן, הפחמן הדו-חמצני. הייצור של גזים אלה אצל אנשים עם ליקויים האנזים לקטאז לקדם נפיחות בבטן, גזים מוגברת, כאב, בחילות, ו- borborygmi (הבטן מקרקרת)<sup class="xref">7</sup>. רמות גבוהות של לקטוז במערכת העיכול יכול גם להוביל שרפרפים רופף.</p>
<p class="jove_content">השליטה של ביטוי גנים <em>LCT</em> הוא מווסת על ידי פולימורפיזמים ממוקם ב אינטרונים של הגן <em>MCM6</em> הסמוך. אנשים עם ביטוי מתמשכת של לקטאז לשאת פולימורפיזמים המתפקדים חזקה דיסטלי מרחיבי <em>LCT</em> ביטוי גנים, ובכך קיזוז הרגיל למטה רגולציה של שעתוק <em>LCT</em> במהלך הגמילה, וכתוצאה מכך ביטוי לקטאז מתפעל לבגרות<sup class="xref">3</sup>. משפר פולימורפיזמים שהוצעו חיובי שבחרו בעקבות הביות של בקר וגמלים במזרח התיכון לפני חמשת אלפים שנה<sup class="xref">9</sup><sup>,</sup><sup class="xref">10</sup>.</p>
<p class="jove_content">ניתן לנהל את הסימפטומים הנובעים ATH על-ידי הפחתת צריכת חלב (לקטוז), לדוגמה על-ידי הסרת מוצרי חלב בתזונה. גישה חלופית עבור ATH, הגישה של בחירה עבור המחלקה לחקירות, הוא השימוש של תוספי לקטאז, זמין נרחב מבתי מרקחת. תוספות אלה מספקים לקטאז מבודד ממגוון רחב של מקורות, כולל שמרים וחיידקים, בצורה נוזלית או מבוסס-הגלולה שניתן לקחת עם או להוסיף מזון המכיל לקטוז. תוספת hydrolyze שיעור של לקטוז נוכח המזון למוצרים גלוקוז, גלקטוז, ובכך יאפשר לקליטתם ומניעת הצטברות של לקטוז מעוכל המצע בבטן.</p>
<p class="jove_content">בהתבסס על השימוש של תוספי לקטאז כסיוע תזונתיים, פיתחנו ניסוי מעבדה אנזימולוגיה פשוט מתאים לתלמידי השנה הראשונה ביו המדע או בית מרקחת. בניסוי מעבדה מנצל תוספי לקטאז זמינים מסחרית, והשימושים אורתופדיה-nitrophenol -<em>beta</em>-D-galactopyranoside (ONPG) כדי לספק נקודת סיום ערכי צבע מוחלטים למדידת המחשוף של איגרות חוב glycosidic על ידי לקטאז (<strong> איור 3</strong>)<sup class="xref">11</sup>. ONPG פועל על סובסטרט מלאכותי עבור? לקטאז, אשר כאשר נתון הידרוליזה על ידי אנזים זה מייצר D-גלקטוז אורתופדיה-nitrophenol. המוצר האחרון בעל צבע צהוב, קליטת אור באורך-גל של 420 ננומטר. מאת לכימות שינויים כלשהם ספיגת-420 nm הבאים חשיפת ONPG לקטאז, זה אפשרי להעריך את הפעילות של אנזים זה. בניסוי מעבדה מספק הפגנה של פעילות ההידרולז אנזימטיות. על ידי בבניין משכפל נוסף, בביצוע מבחני בתנאים שונים, זה ניתן לשלב ניתוח מתוחכמות יותר של קינטיקה אנזימטית, מתן דוגמה ממשית ערך של אנזימים בפעולה הרלוונטיים לבריאות האדם.</p>

<table><tbody><tr><td>Lactase tablet</td><td>Lamberts</td><td>8511-60</td><td></td></tr><tr><td>Phosphate buffered saline (powdered)</td><td>Sigma</td><td>P3813</td><td>Dissolved in deionized water to a final concentration of 100 mM</td></tr><tr><td>ONPG</td><td>Sigma</td><td>N1127</td><td>Dissolved in deionized water to a final concentration of 5 mM</td></tr><tr><td>Sodium Carbonate</td><td>Sigma</td><td>451614</td><td>Dissolved in deionized water to a final concentration of 1 M</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Pestle and mortar</td><td>VWR</td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Jenway 6315</td><td></td><td></td></tr><tr><td>Pipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>15 mL tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Spectrophotometer cuvettes</td><td>Jenway</td><td></td><td></td></tr><tr><td>Vortex</td><td>Vortex genie 2</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Ice bucket</td><td>VWR</td><td></td><td></td></tr><tr><td>Water bath</td><td>Thermo-Scientific</td><td></td><td></td></tr><tr><td>Weighing scales</td><td>Thermo-Scientific</td><td></td><td></td></tr></tbody></table>

measuring lactase enzymatic activity in the teaching lab

<p class="jove_content">להבין איך עובדים אנזימים, הנוגעים זה דוגמאות בחיים האמיתיים, הינה קריטית מגוון רחב של דרגות לתואר ראשון במדעי ביולוגי וביו. זה קל לעקוב אחר הפרוטוקול פותחה עבור סטודנטים לתואר ראשון בית מרקחת בשנה הראשונה ומספק מבוא ברמה התחלתית אנזים תגובות ונהלים אנליטי עבור ניתוח האנזים. האנזים של הבחירה היא? לקטאז, כמו זה מייצג דוגמה של אנזים זמינים מסחרית הרלוונטיים לאימון המחלה האנושית/פרמצבטיקה. לקטאז מופק תוסף תזונה טבליות, העריך שימוש assay ערכי צבע מוחלטים בהתבסס על הידרוליזה של סובסטרט מלאכותי של לקטאז (אורתופדיה-nitrophenol -<em>beta</em>-D-galactopyranoside, ONPG). שחרורו של אורתופדיה-nitrophenol בעקבות את המחשוף hydrolytic של ONPG על ידי לקטאז נמדד על ידי שינוי ספיגת-420 ננומטר, ואת השפעת הטמפרטורה על התגובה אנזימטי מוערך על-ידי ביצוע התגובה על קרח, בטמפרטורת החדר ו- 37 מעלות צלזיוס. ניתוח מתקדם יותר ניתן ליישום בעזרת פרוטוקול זה על ידי הערכת הפעילות אנזים בתנאים שונים באמצעות נוגדנים שונים.</p>

<p class="jove_content" fo:keep-together.within-page="1">נציג תוצאות בסעיף 2 של הפרוטוקול מוצגים <strong class="xfig">באיור 4A</strong>. התגובה שליטה, בהיעדרו של האנזים לקטאז, נשארת ברורה בזמן הפתרון התגובה, המכיל תמצית מן הלוח? לקטאז, הופך צהוב כמו ONPG hydrolyzed שחרור <em>אורתופדיה</em>- nitrophenol. <strong class="xfig">איור 4B</strong> מראה כימות באמצעות יחידות שרירותי של סעיף 4 של הפרוטוקול, הניתוח הבא דוגמאות שימוש ספקטרופוטומטרים. הייצור של אורתופדיה-nitrophenol הוא כאשר התגובה מודגרת על קרח, הגבוהים והנמוכים ביותר כאשר התגובה מתבצע ב- 37 מעלות צלזיוס.</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54377/54377fig1.jpg"><br><strong class="xfig">איור 1</strong> <strong>:</strong> <em><strong>E. coli </strong></em> <strong>בטא-galactosidase.</strong> (<strong>א</strong>) מבנה גבישי של בטא-galactosidase מ- <em>Escherichia coli</em>, מציג את המבנה tetrameric של הפעילים מורכבים. Allolactose (<strong>B</strong>) (מוצג באדום) מאוגדים האתר הפעיל של בטא-galactosidase. תמונות שנוצרו PDB 1JZ8<sup class="xref">4</sup>. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig1large.jpg" target="_blank">אנא לחץ כאן כדי להציג גירסה גדולה יותר של הדמות הזאת.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54377/54377fig2.jpg"><br><strong class="xfig">איור 2</strong> <strong>:</strong> <strong>פעולה אנזימטי של לקטאז.</strong> הידרוליזה של לקטוז על ידי לקטאז לייצר beta-D-גלקטוז, beta-D-גלוקוז. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig2large.jpg" target="_blank">אנא לחץ כאן כדי להציג גירסה גדולה יותר של הדמות הזאת.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54377/54377fig3.jpg"><br><strong class="xfig">איור 3</strong> <strong>:</strong> <strong>הידרוליזה מאורתופדיה-nitrophenol -</strong> <em><strong>בטא</strong></em> <strong>-D-galactopyranoside.</strong> ONPG הידרוליזה על ידי לקטאז לייצר beta-D-גלקטוז, אורתופדיה-nitrophenol (שהוא בצבע צהוב), המאפשר להערכת הפעילות לקטאז על ידי מדידה של ספיגת-420 ננומטר. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig3large.jpg" target="_blank">אנא לחץ כאן כדי להציג גירסה גדולה יותר של הדמות הזאת.</a> </p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54377/54377fig4.jpg"><br><strong class="xfig">איור 4</strong> <strong>:</strong> <strong>הידרוליזה ONPG.</strong> התוצאות נציג של הידרוליזה ONPG, מציג צינורות שליטה ותגובה (<strong>A</strong>), נציג נתונים בסעיף 4 של הפרוטוקול שנרשם בגליונות ב- class, מציג raw ספיגת ביחידות שרירותי-420 nm, מתוקנות על רקע ו בטמפרטורות שלוש שונות (<strong>B</strong>). <a href="https://www.jove.com/files/ftp_upload/54377/54377fig4large.jpg" target="_blank">אנא לחץ כאן כדי להציג גירסה גדולה יותר של הדמות הזאת.</a></p>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td><strong>תנאי ניסיוני</strong></td>
			<td><strong>משתני הניסוי</strong></td>
		</tr>
<tr>
<td>טמפרטורה</td>
			<td>0 ° צלזיוס, 10 ° C, 20 ° C, 30 מעלות צלזיוס, 50 ° C</td>
		</tr>
<tr>
<td>ריכוז המצע</td>
			<td>מ מ 0, 1 מ"מ, 5 מ"מ, 10 מ מ ONPG</td>
		</tr>
<tr>
<td>עיכוב תחרותי</td>
			<td>לקטוז ONPG פלוס מ מ 0, 1 מ"מ, 5 מ"מ או 10 מ מ 5 מ מ</td>
		</tr>
<tr>
<td>התלות בזמן</td>
			<td>0 s, 15 s, 30 s, 1 דקות, 5 דקות, 10 דקות, 20 דקות</td>
		</tr>
<tr>
<td>דנטורציה בחום</td>
			<td>תמצית לקטאז מחומם ל 100 מעלות צלזיוס במשך 10 דקות לפני כדי assay</td>
		</tr>
</tbody></table><p class="jove_content" fo:keep-together.within-page="1"><strong>טבלה 1: פוטנציאל מורחב התנאי ניסיוני</strong>. הציע ניסויים מורחבת, יבוצע שהפקידים, חוקרת היבטים ספציפיים של לקטאז הביולוגיה.</p>

Watch this Scientific Journal Video about Measuring Lactase Enzymatic Activity in the Teaching Lab at JoVE.com

Measuring Lactase Enzymatic Activity in the Teaching Lab

<p class="jove_content">הפעילות האנזימטית מתרחשת של לקטאז חיוני קטבולי עיבוד דו-סוכר חלב (לקטוז). כאן, הפעילות של לקטאז נמצאו ב תוספי מזון הוא לבדיקה באמצעות וזמינותו של ערכי צבע מוחלטים. הדבר מספק לתלמידים פלטפורמה ניסיוני להבנת הפעילות של קינטיקה לקטאז, אנזים.</p>

מדידת פעילות אנזימטי לקטאז במעבדת ההוראה

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

measuring-lactase-enzymatic-activity-in-the-teaching-lab

Education

Research

JoVE Core

JoVE Journal

Cell Biology

Biology

Analytical Chemistry

Anatomy and Physiology

Unit 1

Biochemistry of the Cell

Fundamentals of Chemistry

Energy and Catalysis

Chemical Applications of Statistical Analyses

SCIENTISTS IN ACTION

112.9K Views.  University of Reading. הפעילות האנזימטית מתרחשת של לקטאז חיוני קטבולי עיבוד דו-סוכר חלב (לקטוז). כאן, הפעילות של לקטאז נמצאו ב תוספי מזון הוא לבדיקה באמצעות וזמינותו של ערכי צבע מוחלטים. הדבר מספק לתלמידים פלטפורמה ניסיוני להבנת הפעילות של קינטיקה לקטאז, אנזים.

Video: Measuring Lactase Enzymatic Activity in the Teaching Lab

Lab Techniques

<h4>Measurement Accuracy</h4>
<p>The ability to repeat an experiment and get the same results, or <em>reproducibility</em>, is essential in scientific research. However, it&#8217;s impossible to repeat an experiment if you don&#8217;t know how you did it. Thus, scientists keep detailed records of their experiments in lab notebooks. These records include important information like the amount of each material used and descriptions of each step of the procedure.</p>
<p>Equipment, like balances and volumetric or graduated glassware, is used to measure solid and liquid compounds for experiments. The precision of each measurement is limited by the equipment used. Balances and other equipment with digital readouts automatically display values at the maximum precision that they can support. The precision of graduated glassware and rulers depends on the increment, which is the amount represented by the smallest distance between two marks.</p>
<p>The surface of most liquids curves upwards at the edges in glass containers. This type of curved liquid surface is called a concave meniscus. When the edges of the liquid curve down, it is called a convex meniscus. When measuring liquid volumes, the reference point for the top of the liquid is the bottom of a concave meniscus or the top of a convex meniscus. To accurately identify the position of the meniscus, liquid volumes are read by looking at the liquid&#8217;s surface from the side at eye-level. Looking at the liquid from above or below will cause the liquid surface level to appear higher or lower than it actually is.</p>
<p>When the reference point of the meniscus touches a mark on graduated or volumetric glassware, the glassware is holding the volume defined for that mark. Volumetric glassware is designed to measure a specific volume, so it only has one mark. Graduated glassware is designed to measure a range of volumes, so it has many marks, or <em>graduations</em>, which are labeled at regular intervals. Both graduated and volumetric glassware may have additional manufacturer-provided information about their precision.</p>
<h4>Calibration and Uncertainty</h4>
<p>Manufacturers calibrate equipment to ensure that the measurements are accurate to within a specified range of uncertainty. For example, a high-precision analytical balance may read out to four decimal places (0.0000 g) with an uncertainty of &#177; 0.0001 g. A reading of 0.0345 g indicates that the true value is between 0.0344 g and 0.0346 g.</p>
<p>Glassware typically has its measurement uncertainty printed on it. To accurately record volume measurements, you must consider both the increment and the uncertainty. For example, a precise volume measurement in a 100 mL graduated cylinder with 1 mL increments and an uncertainty of &#177; 0.5 mL would be recorded to the tenths place (000.0 mL &#177; 0.5 mL). If the reference point of the meniscus falls between two marks, you should estimate a value for the tenths place; otherwise, leave it as 0. Since temperature affects volume, the temperature for which the glassware is calibrated will be printed on it.</p>
<p>Glassware is calibrated either <em>to contain</em> (TC) or <em>to deliver</em> (TD). TC glassware holds the specified volume of liquid when it is filled to the mark, but a small amount of liquid will be left behind when it is poured into another container. Solutions are often prepared in TC glassware because the accuracy of the solution volume affects the accuracy of the concentration. Volumetric flasks are typically calibrated to contain.</p>
<p>TD glassware holds a little more than the amount of liquid specified when it is filled to the mark, but it dispenses only the specified volume. Thus, TD glassware should not be emptied completely when dispensing liquids. This type of glassware is useful for transferring a precise volume of liquid to another container. Both graduated and volumetric pipettes are typically calibrated to deliver.</p>
<p>Unless otherwise specified, the uncertainty of a number is assumed to be on the scale of the last digit. Thus, it&#8217;s particularly important to report the uncertainty of a measurement if the uncertainty is on a larger scale. For rulers and graduated glassware without a known uncertainty, the uncertainty is estimated as half of the smallest increment. The uncertain digit is based on where the meniscus falls between two tick marks.</p>
<h4>Significant Figures</h4>
<p>Significant figures are the numbers in a value that are <em>meaningful</em>, or essential for expressing that value with the appropriate precision. All numbers other than leading zeroes (<strong>0.00</strong>1), trailing zeroes (1,<strong>000</strong>), or scientific notation multipliers (<strong>10</strong><sup>x</sup>) are always significant.</p>
<p>Leading zeroes are never significant because they can be removed by rewriting the number either in scientific notation or, if it is an SI unit, in smaller units. For example, there are only three significant figures in the value 0.00123 m because it can be rewritten as either 1.23 &#215; 10<sup>-3</sup> m or 1.23 mm without losing any information. Note that the scaling factor between different multiples of the same SI unit does <em>not</em> affect the number of significant figures.</p>
<p>Trailing zeroes are significant when they are before or after a decimal point. For example, the measurement 100.110 mL has six significant figures and 100.0 mL has four. Trailing zeroes in a number written without a decimal point are assumed to be insignificant unless they are marked otherwise. There are several different conventions for marking significant zeroes in those cases, but to avoid ambiguity it is advisable to rewrite those values in scientific notation. If the measurement is in SI units, use larger units.</p>
<p>The precision of a measurement limits the precision of any values calculated from it. A calculated value has the same number of significant figures as the <em>least</em> precise measurement or value used in its calculation. If the result has too many significant figures, it is rounded to the appropriate precision. If the result has too few significant figures, it is extended with significant trailing zeroes to preserve the precision. The significant figures of experimentally derived values, like molar mass, density, or some unit conversion factors, must also be considered when they are used in calculations.</p>
<p>The number of significant figures in a calculated value is <em>not</em> limited by physical or mathematical constants in formulas or exact numbers, like the number of data points acquired in an experiment. For example, the formula for calculating the volume of a sphere is V = 4/3&#960;r<sup>3</sup>. The number of significant figures in the calculated volume is <em>only</em> affected by the number of significant figures in the radius measurement <strong>r</strong>. 4/3&#8201; and &#960; are constants, and <sup>3</sup> is simply notation for a mathematical operation.</p>
<p>Defined conversion factors are also treated as constants. For instance, the inch is defined as exactly 25.4 millimeters; therefore, the number of significant figures of the converted value depends only on the original measurement. Always check whether the conversion factor is exactly defined or an experimental value when converting between units.</p>
<p>For simple addition and subtraction of measurements, we use significance arithmetic to determine the number of significant figures. In this case, the answer has as many decimal places as the measurement with the fewest decimal places <em>regardless of the number of significant figures</em>. Thus, 15,643.7 mL + 0.613 mL = 15,644.3 mL.</p>
<p>For multi-step calculations or complex equations that you break into smaller parts to solve, you should keep at least one or two insignificant figures on the intermediate values. For example, if you calculate the values of the numerator and denominator of a fraction separately before dividing them, you should retain the insignificant figures of the numerator and denominator when you divide them. This minimizes <em>rounding error</em>, which is the difference from the number you would get if you entered the entire formula into your calculator at once. If you write down these intermediate values, you should note which digits are insignificant.</p>
<h4>References</h4>
<p>Harris, D.C. (2015). <em>Quantitative Chemical Analysis</em>. New York, NY: W.H. Freeman and Company.</p>

Accuracy, Precision, Uncertainty, and Significant Figures - Concept

טכניקות מעבדה

Measurement Accuracy
The ability to repeat an experiment and get the same results, or reproducibility, is essential in scientific research. However, ...

JoVE Lab Manual

Chemistry

Scientific Measurement and Lab Skills

<p><strong>Two-Dimensional Graphs</strong></p>
<p>Some chemistry experiments take the form of directly changing one property of the system that you are studying otherwise known as the independent variable, like temperature, and measuring the effects on another property, otherwise known as the dependent variable, like volume. Once the data have been collected, the interaction between the two parameters must be <i>quantified</i> &#8212; or converted into a form that can be evaluated &#8212; and compared to other relationships.</p>
<p>Two-dimensional graphs can be used to derive certain types of mathematical relationships between two properties or to establish that no such relationship exists between them. The analysis will ultimately determine how the dependent variable changes in response to the independent variable. In the example of adjusting the temperature of a liquid or gas and monitoring changes in its volume, the temperature is the independent variable, and the volume is the dependent variable.</p>
<p>To make a two-dimensional graph, each data point must have a value known as the coordinate for both the dependent and independent variables. The independent variable is plotted on the x-axis, and the dependent variable is plotted on the y-axis. These plots are easily made in spreadsheet software, which can also be used to analyze the plotted data.</p>
<p><strong>Curve Fitting</strong></p>
<p>Once a data set has been plotted on a two-dimensional graph, curve fitting can be used to generate an equation, or <i>function</i>, for the dependent variable in terms of the independent variable. Functions represent a mathematical model that best models the data from which it is derived. Curve fitting is the technique of finding a function that produces a line that is a good match for the pattern of data points. Spreadsheet software has various curve-fitting tools, which is referred to as &#8216;best-fit&#8217;. This is usually a linear least-squares regression analysis, though most software also offers nonlinear least-squares regression.</p>
<p>The precision of the best-fit linear equation can be checked by plugging in the x values for the data points and comparing the &#8216;theoretical&#8217; results of the equation to the actual y values of the data points. Spreadsheet software will typically calculate the coefficient of determination (R<sup>2</sup>) value for the function, which shows how well the function matches the data points. The closer the R<sup>2</sup> value is to 1, the better the fit for a linear regression. Other functions have more specialized methods to determine how well the fit of the function is to the data.</p>
<p>Determining the uncertainty of dependent values calculated from the best-fit function would require complicated &#8220;error propagation&#8221; techniques. However, it is possible to calculate uncertainty within the equation in the form of the standard deviation for both the slope and y-intercept of a best-fit function. This is usually performed with a different tool than the one used to generate a two-dimensional plot.</p>
<p><strong>Standard Deviation</strong></p>
<p>The standard deviation describes the amount of variation present in a set of values. The population standard deviation (&#963;) is used when there is data from each member of a finite population, such as the mass of each marble in a bag of marbles. The sample standard deviation (<i>s</i>) is used for all other cases and is the default standard deviation calculation in spreadsheet software.<sup>1</sup> You can assume that &#8216;standard deviation&#8217; refers to the sample standard deviation.</p>
<p>Random measurement error is assumed to follow a roughly &#8216;normal&#8217; distribution, where about 68% of a set of values lie within a range of one standard deviation on either side of the mean, 95% of the values lie within two standard deviations on either side of the mean, and 99.7% of the values lie within three standard deviations on either side of the mean. Thus, the standard deviation is a useful way to describe error and uncertainty.</p>
<p>The equation for the sample standard deviation is:</p>
<p><img src="https://www.jove.com/CDNSource/lm/labs/42/42_Concepts_1.jpg" /></p>
<p>In this equation, N is the number of values; <img src="https://www.jove.com/CDNSource/lm/labs/42/30034eq02.jpg" /> is the average (or mean) of the values; and x<sub>i</sub> represents each individual value. Thus, to calculate the standard deviation by hand, calculate the mean of the set of values, subtract the mean from each value, square each difference, add the squared differences, divide the total sum by one less than the number of values, and take the square root of the quotient. The closer <i>s</i> is to zero, the less variation there is between the values. If the values are entered into spreadsheet software, the standard deviation can be calculated from within the software.</p>
<p>The number of significant figures in a standard deviation depends on what values it is for. When reporting the standard deviation for a group of data points taken under the same conditions, the appropriate number of significant figures in the mean value must first be determined. The standard deviation is then rounded to the same number of decimal places as the mean. For a set of volumes with four significant figures, a mean of 15.361 mL, and a standard deviation of 0.2313, the mean and standard deviation would be reported as 15.36 mL &#177; 0.23 mL.</p>
<p>When reporting the standard deviation of the mean and y-intercept for a best-fit function determined by least-squares analysis, which is the usual method for spreadsheet software, the first decimal place of the standard deviation is the last significant figure of the mean or y-intercept. Thus, the standard deviation should be rounded to one significant decimal place, and the slope or y-intercept should be rounded to the corresponding decimal place. For example, if the slope is 0.1691 L&#183;K<sup>-1</sup> and it has a standard deviation of 0.00512, the slope should be reported as 0.169 L&#183;K<sup>-1</sup> &#177; 0.005 L&#183;K<sup>-1</sup>.</p>
<p>If the standard deviation of the slope or y-intercept is so much smaller than its corresponding value that following this rule would give more significant figures to the slope or y-intercept than the original measurement data would allow, then instead determine the significant figures of the slope or y-intercept from the x and y values and round the standard deviation to one significant figure. Thus, for a slope of 0.1691 L&#183;K<sup>-1</sup>, a standard deviation of 0.0000512, and x and y values with four significant figures, the slope should be reported as 0.1691 L&#183;K<sup>-1</sup> &#177; 0.00005 L&#183;K<sup>-&#8205;1</sup>. In this case, it&#8217;s fine for the calculated value and its standard deviation to have a different number of decimal places. Keep in mind that the least-squares method uses both the x and y values to calculate the slope and y-intercept.</p>
<p>When reporting standard deviation as an uncertainty range, be sure to note specifically that the uncertainty represents one standard deviation. This tells the reader that there is about a 68% chance that the true value of a measurement falls within that range of the mean, assuming a normal distribution. It may often be more suitable to report uncertainty as two standard deviations from the mean, as that increases the probability to about 95%. To do this, simply multiply the standard deviation by two before rounding it to the appropriate number of significant figures.</p>
<p><strong>References</strong></p>
<ol style="font-size:17px">
	<li style="font-size:17px">Harris, D.C. (2015). <i>Quantitative Chemical Analysis</i>. New York, NY: W.H. Freeman and Company.</li>
</ol>

Two-Dimensional Graphs, Curve Fitting, and Standard Deviation - Concept

מדידה מדעית ומיומנויות מעבדה

Two-Dimensional Graphs
Some chemistry experiments take the form of directly changing one property of the system that you are studying otherwise known as ...

Acid and Base Concentrations

<h4>Acids and Bases</h4>
<p>An Arrhenius acid produces hydrogen ions when it is dissolved in water:</p>
<p style="text-align: center;">HA + H<sub>2</sub>O &#8594; H<sup>+</sup><sub>(aq)</sub> + A<sup>-</sup><sub>(aq)</sub></p>
<p>Here, HA is the non-dissociated acid, H<sup>+</sup> is the hydrogen cation, and A<sup>-</sup> is the solvated anion &#8212; called the conjugate base. An Arrhenius base produces hydroxide ions when dissolved in water:</p>
<p style="text-align: center;">BOH + H<sub>2</sub>O &#8594; B<sup>+</sup><sub>(aq)</sub> + OH<sup>-</sup><sub>(aq)</sub></p>
<p>Here, BOH is the non-dissociated base, OH<sup>-</sup> is the hydroxide ion, and B<sup>+</sup> is the solvated cation &#8212; called the conjugate acid. A conjugate base is formed when an acid loses a hydrogen ion and has the potential to gain a hydrogen. The same follows for a conjugate acid, which is formed when a base loses a hydroxyl group and has the potential to regain it. Every acid has a conjugate base, and every base has a conjugate acid.</p>
<h4>pH</h4>
<p>pH is the degree of acidity of the solution and is a measure of the amount of hydrogen ions in a solution. The pH scale is logarithmic and runs from 0 to 14; aqueous solutions with a pH below 7 are described as acidic, and aqueous solutions with a pH above 7 are described as alkaline or basic. Solutions at pH 7 are considered neutral.</p>
<p>The pH of a solution is equal to the negative log base ten of the concentration of hydrogen ions in solution.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_3.jpg" /></p>
<p>Water interacts strongly with the hydrogen ion because its strong positive charge attracts the negative pole of surrounding water molecules. In fact, they interact so strongly that they form a covalent bond and the H<sub>3</sub>O<sup>+</sup> cation, called hydronium. The above equation is rewritten to reflect this.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_4.jpg" /></p>
<p>For simplicity, we&#8217;ll refer to the concentration of hydrogen ions instead of hydronium ions when discussing pH. The lower the pH value of a solution, the more hydrogen ions that are present, and by extension, the more acidic the solution. For example, the pH of 1 mM of sulfuric acid is 2.75, whereas the pH of 1 mM of hydrochloric is 3.01. The concentration of hydrogen ions in the sulfuric acid solution is calculated as 1 &#215; 10<sup>-2.75</sup>, whereas the concentration of hydrogen ions in the hydrochloric acid solution is 1 &#215; 10<sup>-3.01</sup>. Thus, there are more hydrogen ions present in sulfuric acid, and it is more acidic. Remember, even though the pH of two solutions may vary by as little as half a pH value, due to the logarithmic nature of the pH scale, the amount of hydrogen varies greatly.</p>
<h4>Strength of Acids and Bases</h4>
<p>An acid&#8217;s strength is affected by the electronegativity of the conjugate base and the polarity of the acidic hydrogen. Strength, therefore, refers to how readily the hydrogen cation (H<sup>+</sup>) disassociates from the anion. Strong acids and bases dissociate entirely in aqueous solutions, whereas weak acids and bases only dissociate partially into their conjugate ions.</p>
<p>The dissociation constant, K<sub>a</sub>, represents acid strength. K<sub>a </sub>is calculated using the concentrations of the non-dissociated acid HA, and the concentrations of the hydrogen cations and the conjugate base, A<sup>-</sup>. Higher K<sub>a</sub> values represent stronger acids, whereas smaller K<sub>a</sub> values represent weaker acids.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_5.jpg" /></p>
<p>K<sub>a</sub> is numerically very small, and it is often reported in the form of pK<sub>a</sub>, which is the negative log base ten of K<sub>a</sub>. Lower pK<sub>a</sub> values correspond to a stronger acid, whereas higher pK<sub>a</sub> values correspond to a weaker acid.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_6.jpg" /></p>
<p>Some acids dissociate only one hydrogen ion and therefore have one pK<sub>a</sub> value. These acids are called monoprotic. However, some acids can dissociate more than one hydrogen ion and are called polyprotic. These acids have a pK<sub>a</sub> value for each hydrogen ion dissociation.</p>
<p>pK<sub>a</sub> can also be used to calculate the equilibrium pH of an acid-base reaction, as shown in the Henderson-Hasselbalch equation.</p>
<p style="text-align: center;"><img src="https://www.jove.com/CDNSource/lm/labs/44/44_Concepts_7.jpg" /></p>
<p>The Henderson-Hasselbalch equation is used to calculate pH, when the concentrations of the conjugate base and the weak acid are known, or to calculate the pK<sub>a</sub> if the pH and concentrations are known.</p>
<h4>Titration</h4>
<p>Acid-base reactions are quantitatively studied using titration. In a titration experiment, a solution of a known concentration, called a standard solution, is used to determine the concentration of another solution. For acid-base titrations, a standardized solution of base is slowly added to an acid of unknown concentration (or the acid is added to the base). The acid-base reaction is a neutralization reaction, which forms a salt and water. When the moles of hydrogen ions in the acid are equal to the moles of hydroxyl ions added from the base, the solution reaches neutral pH.</p>
<p>To perform an acid-base titration, the standardized base is slowly added to a stirring flask of the unknown acid using a burette, which enables the measurement of volume and the dropwise addition of base. The pH of the solution is closely monitored throughout the titration using a pH indicator added to the acid. Typically, phenolphthalein is used as the solution remains colorless until it becomes basic, turning a light pink.</p>
<p>As the titration approaches the equivalence point, which is when the moles of hydrogen ions equal the moles of hydroxyl ions added, the pH indicator temporarily changes color due to an excess of hydroxyl ions. When the flask is swirled, the pH indicator&#8217;s acidic color returns. The titration is complete and has reached its endpoint when a tiny excess of hydroxyl ions changes the indicator permanently to its basic color.</p>
<p>The titration curve is a plot of the pH of a solution versus the volume of standardized base added. The equivalence point is located at the inflection point of the curve, and it is calculated as the second derivative of the titration curve.</p>
<p>If an acid is polyprotic, it will have multiple equivalence points, one for each hydrogen ion dissociation. The pH at the halfway point to the equivalence point for monoprotic acids, or between equivalence points in the case of polyprotic acids, is equal to the pK<sub>a</sub> of the acid.</p>
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). <em>Chemistry and Chemical Reactivity.</em> Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silberberg, M.S. (2009). <em>Chemistry: The Molecular Nature of Matter and Change.</em> Boston, MA: McGraw-Hill.</li>
	<li style="font-size:17px">Harris, D.C. (2015). <em>Quantitative Chemical Analysis.</em> New York, NY: W.H. Freeman and Company.</li>
</ol>

Acid-Base Titration: Calculating pH, Strength, and Concentration - Concept

ריכוזי חומצה ובסיס

Acids and Bases
An Arrhenius acid produces hydrogen ions when it is dissolved in water:
HA + H2O &#8594; H+(aq) + A-(aq)
Here, HA is the non-dissociated ...

Esterification

<h4><strong>Esters</strong></h4>
<p>The structure of an ester is a carbonyl with an alkyl or aryl group (R) on one side, and an oxygen bound to another alkyl or aryl group (R&#8217;) on the other side, represented by the general formula: RCOOR&#8217;. Esters can be commonly derived by an esterification reaction between a carboxylic acid and an alcohol. The hydroxyl group of the carboxylic acid is replaced by an alkyl or aryl group. Simple esters, which have low molecular weight and small R and R&#8217; functional groups, have smells and flavors of fruits and flowers.</p>
<p>The simplest carboxylic acid is formic acid (HCOOH), with the simplest alcohol being methanol. Thus, the simplest ester formed from the esterification of these two molecules is methyl formate (HCOOCH<sub>3</sub>). Methyl formate is considered a derivative of formic acid because the OH group is replaced by the OCH<sub>3</sub> group. The structural differences between methyl formate and formic acid dictate the difference in their properties. This is because the properties of the molecule depend heavily on the R and R&#8217; groups.</p>
<p>Esters that are found in fruits and flowers are known as simple esters due to the low complexity of their structure in comparison to other esters. The alkyl and aryl groups found on these esters greatly influences the fragrance they emit; thus, the addition of a carbon or a different functional group can impact the scent produced by the ester. For example, propyl acetate (C<sub>5</sub>H<sub>10</sub>O<sub>2</sub>) has the scent of pears. However, butyl acetate (C<sub>6</sub>H<sub>12</sub>O<sub>2</sub>), which has only one more carbon in its chain, smells like apples.</p>
<p>Long-chain esters, meaning those with long alkyl chains, are called fatty acids. Fatty acids are important biological compounds that are essential for certain biological processes. Fatty acids are also a biological form of energy storage. When three fatty acids react with glycerol, they form a triglyceride &#8212; the most common form of fatty acids. Triglycerides have a very high molecular weight and can be highly branched. Their function and properties are dependent on the length and types of alkyl chains incorporated into their structure. One of the most common industrial uses for triglycerides is in the production of soaps and detergents.</p>
<h4><strong>Esterification Reaction</strong></h4>
<p>Esters are formed from an esterification reaction, with simple esters being formed through Fisher esterification. This reaction converts a carboxylic acid and alcohol into an ester with water as a by-product. Fisher esterification is a reversible reaction that proceeds very slowly. An acid catalyst, typically in the form of sulfuric acid, is added to increase the rate of the reaction while also acting as a dehydrating agent.</p>
<p>The mechanism of action for a Fisher esterification begins by the carbonyl oxygen attacking the sulfuric acid and deprotonating its OH group. This leads to a positively charged carbonyl oxygen. To better stabilize the positive charge, one electron pair of the carbonyl double bond is pushed to the carbonyl oxygen while simultaneously creating an electron-poor carbon center. Next, the hydroxy group of the alcohol acts as a nucleophile and attacks the electron-poor carbon &#8212; the electrophile &#8212; forming an intermediate. Then, the positively-charged hydroxyl group from the alcohol is deprotonated by the hydroxyl group of the carboxylic acid, stabilizing the oxygen atom of the alcohol. In the last step, the carbonyl oxygen is deprotonated by the conjugated base, and the free electron pair is moved towards the carbonyl center while the protonated carboxyl group leaves as a water molecule.</p>
<p>Since the esterification reaction is reversible, a 1:1 mixture of the carboxylic acid and the alcohol will reach equilibrium with about 70% yield of the ester. However, as with any equilibrium process, the reaction can be driven in one direction by changing the concentration, pressure, temperature, or volume of the reagents. This change causes the reaction to adjust to a new equilibrium to counteract the change, which is the basis of Le Chatelier&#8217;s principle.</p>
<p>When the concentration of one of the reactants is increased, the equilibrium shifts in the direction that will decrease its concentration. Thus, by increasing the concentration of one reactant, the equilibrium produces more of the product, thereby resulting in a higher yield of the ester product.</p>
<p>One common method utilized in organic chemistry labs is to provide an excess of one of the reagents, typically the alcohol. As mentioned earlier, Fisher esterification can utilize sulfuric acid as a catalyst. However, one of the other functions of sulfuric acid is to act as a dehydrating agent, sequestering water molecules away from the reaction. Applying Le Chatelier&#8217;s principle demonstrates that as one of the products is removed &#8212; in this case, water &#8212; the reaction is pushed to making more product. This increases the yield of the ester formed.</p>
<h4><strong>Reference</strong></h4>
<ol>
	<li style="font-size:17px">Streitwieser, A., Heathcock, C.H., Kosower, E.M. (1998). <em>Introduction to Organic Chemistry. </em>Upper Saddle River, NJ: Prentice Hall.</li>
</ol>

Esterification: Synthesis of Ester by Fischer Esterification - Concept

אסטריפיקציה

Esters
The structure of an ester is a carbonyl with an alkyl or aryl group (R) on one side, and an oxygen bound to another alkyl or aryl group (R&#8217;) ...

Enzyme Activity

<h4>Biological Catalysts</h4>

<p>All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require an input of energy to start, which is called the activation energy. Catalysts are chemicals that lower the activation energy. Even though catalysts facilitate a chemical reaction, they are not consumed by it. This means a catalyst can facilitate a specific chemical reaction over and over in succession, increasing the rate of that reaction.</p>

<p>Enzymes are biological catalysts that increase the speed and efficiency of biochemical reactions. Most enzymes are proteins but certain ribonucleic acid molecules also have catalytic properties. Each type of enzyme can only bind to one or a few specific substrates, therefore, there is a vast diversity of enzymes, all with distinct functions. The specific reaction of an enzyme depends on its active sites, which has shapes that closely matches the shape of its substrates. Upon substrate binding, the enzyme changes shape slightly in a way that allows the chemical reaction to happen more readily, thus reducing the activation energy of the reaction. Once the reaction takes place, the enzyme releases the products and returns to its original shape, which allows it to repeat the process again.</p>

<h4>Catabolytic versus Anabolic Enzymes</h4>

<p>Most enzymatic reactions can be categorized as either catabolic and anabolic, which have opposite functions. Enzymes that mediate catabolic reactions break down larger substances into multiple products. A well-known catabolic enzyme is lactase, which splits the disaccharide lactose in dairy products into monosaccharides galactose and glucose. Humans have large amounts of lactase in their small intestine after birth, however many individuals lose more than 90% of their lactase by early childhood<sup>1</sup>. This reduction diminishes the ability to digest lactose in an age-dependent manner, which is known as lactose intolerance<sup>2</sup>.</p>

<p>Anabolic enzymes combine multiple substances into a single product. For example, the enzyme DNA polymerase synthesizes DNA molecules by joining nucleotides together. Similarly, the enzyme glycogen synthase connects glucose molecules to generate glycogen. Insulin regulates the activity of glycogen synthase, therefore diabetic patients with low to no insulin levels cannot synthesize glycogen without insulin treatment<sup>3</sup>.</p>

<h4>Environmental Factors</h4>

<p>The shape and charge of the active site of an enzyme is tailored to the specific chemical reaction it catalyzes, therefore the enzyme activity is influenced by factors that affect its active site. Hence, enzymes require specific conditions to work at optimal levels and depending on their function and location, these conditions can vary greatly. These factors include pH and temperature. For example, active sites of enzymes often contain acidic or basic amino acids. Deviations from the optimum pH alter the charges in the active site, making it difficult for the substrates to interact with the enzyme. This property has been used for centuries to preserve food by pickling, which reduces the pH of the pickled food item to a level that effectively inhibits microbial enzymatic activity. Similarly, each enzyme works at an optimal temperature range. If the temperature shifts below or above this range, it alters the shape of the active site, preventing it from effectively interacting with its substrates. In most cases, returning to the optimal temperature restores the enzyme&#8217;s original shape and function, however, temperatures that are too high can denature or irreversibly damage the enzyme structure. Again, these properties are useful for food safety; we cook meat thoroughly to kill harmful bacteria by denaturing their enzymes and refrigerate most food items to slow down enzymatic activity of microorganisms.</p>

<p>Extremophiles, or organisms found in extreme conditions that are lethal to most forms of life, have enzymes capable of functioning at those extremes. For instance, <em>Thermus aquaticus</em> is a bacterium found in hot springs and hydrothermal vents reaching temperatures at which most proteins would denature. Taq polymerase is a thermostable DNA polymerase found in <em>T. aquaticus</em>, which has allowed the development of polymerase chain reaction (PCR), a widely-used laboratory method for rapid DNA replication. PCR requires high temperatures to separate the strands of the DNA double helix to be replicated. Taq polymerase remains stable at these high temperatures while most enzymes would be degraded. PCR cycles between high temperatures that break the DNA into single strands and lower temperatures that allow the polymerase to build new double stranded DNA.</p>

<h4>Molecular Factors that Influence Enzyme Activity</h4>

<p>In addition to specific pH and temperature ranges, some enzymes function better in the presence of other molecules or ions. Coenzymes are biomolecules that bind to the active site of the enzyme and participate in catalysis without getting consumed by the reaction. Coenzymes often transport electrons, specific atoms and functional groups, and thus act as intermediary carriers. B vitamins act as coenzymes for numerous biochemical reactions. For instance, coenzyme A (coA), which is the active form of pantothenic acid, is required by 4% of all mammalian enzymes<sup>4</sup>. Cofactors are often classified as inorganic substances, such as ions, that are required for the enzymatic function. For example, all DNA polymerase enzymes require Mg<sup>2+</sup> or Mn<sup>2+</sup> for DNA replication as well as excision of incorrectly incorporated nucleotides<sup>5</sup>.</p>

<p>There are also other molecules that are not necessary for an enzyme to function but are capable of altering the enzymatic activity. Substances that increase the enzyme activity are called activators, whereas others that decrease the enzyme activity are called inhibitors. These molecules bind to enzymes either at the active site or other locations on the enzyme known as allosteric sites. Sometimes these molecules can bind to the exact location in the active site as the substrate, and thus compete with the substrate for the enzyme binding. Others, especially the substances that bind to allosteric sites, can alter enzyme function without blocking the substrate binding to the active site, and are thus non-competitive. Moreover, binding of inhibitors can be reversible or irreversible. When the interaction is reversable, the enzyme can resume its function once the inhibitor detaches. However, when the binding is irreversible, the inhibitor never detaches from the enzyme and the enzyme becomes irreparably damaged and needs to be replaced. Nevertheless, enzyme inhibitors are useful as well. For instance, inhibitors have been utilized as antibiotics to inhibit bacterial growth, and as chemotherapeutics to inhibit the uncontrolled division of cancer cells<sup>6</sup>.</p>

<h4>Measuring Enzyme Function</h4>

<p>One method used by scientists to study the effects of different conditions on enzyme function is to quantify the reaction rate, which is how fast the enzyme is catalyzing the reaction. Therefore, a higher reaction rate indicates that the enzyme is catalyzing reactions more efficiently, whereas a lower rate means that it is working less efficiently. To determine the reaction rate, the reaction velocity is calculated by measuring the amount of product made per unit time. This is done by measuring the product concentration starting right at the beginning of the reaction when the product concentration is increasing linearly, until the enzyme is completely saturated with the substrate and the catalysis reaches a steady level. Once the baseline reaction rate is determined, it can be used as a reference point, or control, when comparing between a variety of treatments. We can assess reaction rates as relative or absolute measures. A relative reaction rate is a comparative measure that shows differences between two or more conditions, such as treatments in a single experiment, but does not give a value for what is being measured. An absolute reaction rate relies on a quantitative measure of the progress of the reaction. An easy way to understand the difference between relative and absolute reaction rates is to consider measuring the speed of a car in a race. A relative method would compare by assessing the ranking of cars at the finish line, whereas the absolute rate would assess and compare the speed of individual cars in miles per hour.</p>

<p>Since enzymes are so widely used, researchers have begun looking for ways to create and modify enzymes to suit specific needs, known as protein engineering. Recombinant DNA technology allows amino acid sequences to be changed in order to alter an enzyme&#8217;s shape or active site affinity. This has been used to create highly sensitive diagnostic tests for various conditions, as well as for bioremediation efforts and waste management tools<sup>7</sup>.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>Swagerty DL Jr, Walling AD, Klein RM.</strong> Lactose intolerance. <em>Am Fam Physician. </em>2002, Vol. 65, 9: 1845-50.</li>
	<li><strong>Lomer MC, Parkes GC, Sanderson JD.</strong> Review article: lactose intolerance in clinical practice--myths and realities. <em>Aliment Pharmacol Ther. </em>2008, Vol. 27, 2: 93-103.</li>
	<li><strong>AR, Saltiel.</strong> New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. <em>Cell. </em>2001, Vol. 104, 4: 517-29.</li>
	<li><strong>DO, Kennedy.</strong> B Vitamins and the Brain: Mechanisms, Dose and Efficacy--A Review. <em>Nutrients. </em>2016, Vol. 8, 2: 68 doi:10.3390/nu8020068.</li>
	<li><strong>Vashishtha AK, Wang J, Konigsberg WH.</strong> Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases. <em>J Biol Chem. </em>2016, Vol. 291, 40: 20869-20875.</li>
	<li><strong>Watson ID, Stewart MJ, Platt DJ.</strong> Clinical pharmacokinetics of enzyme inhibitors in antimicrobial chemotherapy. <em>Clin Pharmacokinet. </em>1988, Vol. 15, 3: 133-64.</li>
	<li><strong>Ahuja SK, Ferreira, GM, Moreira AR.</strong> Utilization of Enzymes for Environmental Applications. <em>Crit Rev Biotech. </em>2004, Vols. 2-3, 125-54.</li>
</ol>


Enzyme Activity: Effect of pH and Temperature on Peroxidase Activity - Concept

פעילות אנזימים

Biological Catalysts

All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require ...

Cellular Processes

Hydrolysis of an Ester

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you get started, put on a lab coat, safety glasses, and nitrile gloves. Ensure that the lab is equipped with enough lab coats, splash goggles, and gloves for all of the students.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 1 L of 3 M NaOH. Measure 120 g of NaOH pellets, and transfer the pellets to a 1-L volumetric flask. Add deionized water to the 1-L line. Place a stir bar in the flask and mix the solution until the pellets are fully dissolved.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Once the solution is homogeneous, transfer the solution to a glass bottle, cap it, and label it. Place the bottle in a central hood so that students have access to it.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare 1 L of 1 M CaCl<sub>2</sub> solution. Weigh 110.98 g of CaCl<sub>2</sub>, and transfer it to a 1-L volumetric flask. Fill the flask to the marked line with deionized water, add a stir bar, and mix the solution on a stir plate until the CaCl<sub>2</sub> is fully dissolved.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Once the solution is homogeneous, transfer it to a clean glass bottle, cap it, and label it. Store the solution in a central hood.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Prepare at least 1 L of 3 M HCl, which will be used to neutralize any excess sodium hydroxide at the end of the experiment.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Obtain 5 silicone soap molds and leave them in a common area. Obtain at least five different fragrance oils and five powder soap dyes in a variety of colors, and set them near the molds.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place a container of commercial liquid soap in a common area. Set coconut oil next to the balance making sure there are clean spatulas and weighing boats.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;pH meter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL plastic squeeze bottle of DI water</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;50-mL volumetric flasks</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;100-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;6&#160;&#160;&#160;&#160;50-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Glass vials</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small spoon</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small spatula</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Hydrolysis of an Ester: Preparation of Soap - Prep

הידרוליזה של אסטר

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of LaboratoryExpand
	Here, we show the laboratory preparation for ...

Lab Manual Sub Articles

Identification of Unknown Aldehydes and Ketones

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Test Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you start, put on a lab coat, splash-proof safety glasses, and nitrile gloves. <strong>Note: </strong>DNPH is a shock explosive, so handle it gently with low friction tools. Phosphoric acid is also toxic and corrosive.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 100 mL of a 0.1 M solution of DNPH in a 50/50 mixture of 85% phosphoric acid and ethanol. Add a medium stir bar to a 250-mL beaker. In a fume hood, measure 3 g of DNPH and gently pour it into the beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 50 mL of 85% by weight aqueous phosphoric acid and slowly add it to the beaker. Place the mixture on a stir plate and gently stir it until the DNPH dissolves.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure 50 mL of ethanol and pour it into the beaker. Once the solution looks homogeneous, turn off the stir motor and retrieve the stir bar.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Vacuum filter the solution to remove residual solids before transferring it to a 100-mL container. Cap the container, label it &#8216;DNPH solution&#8217;, and place it in a hood for the test reagents.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Prepare 200 mL of iodoform reagent. Measure 50 g of potassium iodide into a clean 250-mL beaker. Add 200 mL of deionized water and stir the mixture on a stir plate until the potassium iodide has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure 25 g of iodine and add it to the stirring solution. Once the iodine has dissolved, turn off the stir motor. Transfer the solution to a labeled 200-mL container and cap it. Place the iodoform reagent in the test solution hood.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Make 22 mL of Tollens&#8217; reagent. Measure 0.6115 g of silver nitrate and pour it into a 50-mL beaker. <strong>Note: </strong>Prepare this solution right before the lab because explosive silver nitride will start to form as the reagent decomposes.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add 12 mL of deionized water and stir until the silver nitrate has dissolved completely to form a 0.3 M silver nitrate solution.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure 6 mL of 3 M NaOH and add it to the stirring silver nitrate solution. Brown silver oxide will precipitate from the solution.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 4 mL of 2 M aqueous ammonia. Use a Pasteur pipette to slowly add it to the stirring solution.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once you have added all 4 mL of ammonia, remove the stir bar and vacuum filter the solution. Pour the filtrate into a 25-mL brown glass bottle.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Wrap the bottle in foil to shield it from light and label it &#8216;Tollens&#39; Reagent&#8217;. Put the wrapped bottle with the other test solutions.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Make sure that the sinks have paper towels and test tube brushes.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Set up appropriate waste containers in a waste collection hood and place glass waste nearby.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Fill a wash bottle with acetone and place it in the waste hood so that students can rinse strong-smelling chemicals out of their Pasteur pipettes before disposing of them.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">In a fume hood, pour about 5 mL of butanone in a labeled vial for each lab group.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Prepare and label vials containing 5 mL of benzaldehyde in the same way. These are the known compounds that every student group will need.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">The unknown compounds are acetophenone, butanol, 3-methyl-2-butanone, pentanol, 2-pentanone, and 3-pentanone. For each compound, prepare two vials containing 5 mL of that compound and label them with a code name. Place the known and unknown vials in a central area.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small 3-prong clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Vacuum line</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube rack</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube tongs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;4&#160;&#160;&#160;&#160;250-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;150-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;5&#160;&#160;&#160;&#160;50-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;5&#160;&#160;&#160;&#160;5-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;B&#252;chner funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Filter adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;4&#160;&#160;&#160;&#160;Small watch glasses</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Thermometer with beaker clip</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;Pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of lab tape and marking pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL bottle of DI water</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Put Pasteur pipettes, filter papers, and small test tubes in a central area.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 3 M NaOH and 1,2-dimethoxyethane in a hood reserved for solvents.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Set up the melting point analyzer and ensure that there are enough capillaries.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">At the end of the lab, promptly dilute any unused Tollens&#39; reagent with at least 20 mL of water and flush it down the drain with copious tap water. <strong>Note: </strong>If necessary, use concentrated hydrochloric acid to precipitate the silver from the Tollens&#39; reagent waste first.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Clean silver-coated test tubes with concentrated nitric acid in a fume hood. Neutralize and dispose of the silver waste according to your lab&#39;s standard procedures.</div>
		</li>
	</ul>
	</li>
</ol>

Test for Aldehydes and Ketones: DNPH, Tollen's Test, and Iodoform Test - Prep

זיהוי אלדהידים וקטונים לא ידועים

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of Test SolutionsExpand
	Here, we show the laboratory preparation ...

Proper Lab Notebook Keeping

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Put on a lab coat, splash-proof safety glasses, and nitrile gloves. You&#39;ll work in a fume hood because these acids are toxic and corrosive.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Ensure that the lab has containers for concentrated acid waste, aqueous acid waste, and glass waste.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Prepare a bottle of concentrated sulfuric acid for each student group. Measure 10 mL of 98% w/w sulfuric acid with a graduated cylinder and pour it into 20-mL bottles using a funnel. Tightly cap the bottles and label them with the solution name and concentration.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Prepare a bottle of nitric acid for each student group. Measure 10 mL of 70% w/w nitric acid with a clean graduated cylinder and pour it into 20-mL bottles via a clean funnel. Cap the bottles and label them with the name and concentration.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Set one bottle of nitric acid and one bottle of sulfuric acid at each hood.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Place a bottle of methyl benzoate in a common hood.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Put Pasteur pipettes and circular filter papers on a central bench.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Confirm that the balance area has enough weighing boats, spatulas, and lab wipes for the class.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Lab stands</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;Small 2-prong clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;10-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;25-mL Erlenmeyer flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;20-mL test tube</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Large B&#252;chner funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Rubber filter adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Thermometer</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Roll of lab tape</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Marker/pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;Small beakers</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

שמירת מחברת מעבדה נכונה

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of the LaboratoryExpand
	Here, we show the laboratory preparation ...

Synthesis of Luminol

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. The chemicals that you will use are hazardous, so both solutions must be prepared and handled in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 10 mL of a 10% by volume solution of hydrazine in water. Measure 8.4 mL of deionized water and add it to a 30-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 1.6 mL of hydrazine hydrate and pour it into the beaker for a total volume of 10 mL. Stir the hydrazine solution with a glass rod until it is well mixed. <strong>Note:</strong> Hydrazine is flammable, toxic, and explosive.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Pour the solution into a 20-mL glass bottle. Cap it and label it with the solution&#39;s name and concentration. Store the hydrazine hydrate and the hydrazine solution in a cabinet for flammables.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare 20 mL of 2.8 to 3 M aqueous NaOH. <strong>Note:</strong> NaOH is highly corrosive. Avoid skin contact.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Measure 2.2 to 2.4 g of solid NaOH and add it to a 50-mL beaker. Add 20 mL of deionized water and stir the mixture on a stir plate until the NaOH has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Pour the solution into a 20-mL glass bottle and close it with a lid. Label the bottle with the solution&#39;s name and approximate concentration. Store the NaOH pellets and solution in the appropriate cabinets.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Lastly, clean your glassware following your usual procedures.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Before you set up the main lab, reserve a separate room that you can make as dark as possible.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Prepare a container for fluorescein waste and ensure that a glass waste container is available.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Set out a 10% sodium hypochlorite (bleach) solution and a 1 M sodium carbonate solution in the waste hood.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Fill a wash bottle with deionized water and place it in the same hood. Confirm that there are paper towels and a test tube brush by each sink in the main lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring containers of 3-nitrophthalic acid, anhydrous sodium dithionite, solid potassium hydroxide, and fluorescein to the balance area.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Confirm that there are enough laboratory wipes, spatulas, and weighing boats for the class.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set triethylene glycol, the hydrazine solution, the NaOH solution, glacial acetic acid, and dimethyl sulfoxide in a central hood. Place a 1-mL graduated pipette and a pipette controller designated for hydrazine in the same hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Put Pasteur pipettes, filter papers, scissors, and plastic paraffin film in a central area.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stirring hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Bunsen burner with tubing</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Lab stands</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;Clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pair of flask tongs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;4&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;25-mL Erlenmeyer flasks</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;250-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;25-mL test tubes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;B&#252;chner funnels</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Long glass stirring rod</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Medium stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Thermometers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Spatulas</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;6&#160;&#160;&#160;&#160;Boiling chips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Medium filter adapter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube stopper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small flask stopper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pair of tweezers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Fire striker or lighter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of laboratory tape or labels</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pen</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Prep

סינתזה של לומינול

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of SolutionsExpand
	Here, we show the laboratory preparation for ...

Beer's Law

Beer-Lambert Law and Equilibrium Constant Determination - Procedure

חוק הבירה

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Lab Setup for Beer&#8217;s LawExpand
	In this lab, you will combine aqueous solutions of FeCl3 ...